Kit for detecting free breast cancer cell marker in blood

Abstract

The invention discloses a kit for detecting a breast cancer marker. The kit comprises the following components: magnetic bead processing solution, a magnetic bead with a breast cancer antibody, 4.0 to 5.0mu l of 10X buffer solution, 4.0 to 5.0mu l of deoxynucleoside triphosphates (dNTPs), 2.0 to 3.0mu l of reverse transcriptase, 0.4 to 0.6mu l of 40u / mu l ribonucleic acid (RNA) enzyme inhibitor, 20.0 to 30.0mu l of 10X hot start polymerase chain reaction (PCR) mixed solution, 10.0 to 15.0mu l of ddH2O and 3.0 to 5.0mu l of breast cancer marker primer. Compared with the prior art, the kit can more accurately detect the breast cancer marker in peripheral blood, and has great guiding significance for selecting breast cancer prognosis schemes and treatment 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 breast cancer cells (malignant tumor cells) from the circulating blood of breast cancer patients, especially capable of separating A detection kit for extracting and identifying free breast cancer cells remaining in the patient's blood after radiotherapy and chemotherapy. Background technique

[0002] As the killer of malignant tumors, breast cancer has an obvious rising trend. The high incidence of breast cancer is increasingly harmful to human beings, especially women. For the early diagnosis and screening of breast cancer and the prognosis of treatment effect, as well as the evaluation and prediction of the curative effect after chemotherapy and radiotherapy, it is of great practical significance to reduce the mortality rate of breast cancer and increase the 5-year survival rate.

[0003] ...

Examples

Embodiment 1

[0038] The preparation of embodiment 1 magnetic beads

[0039] Preparation of antibody immunomagnetic beads: the antibodies used were anti-BerEP4 monoclonal antibody, anti-cytokeratin monoclonal antibody and anti-HER2 monoclonal antibody. In the present invention, the above three kinds of antibodies are labeled separately and then mixed for use. It is also possible to select one or two antibodies for separate labeling and mixed use.

[0040] 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

[0041] The separation of embodiment 2 breast cancer cells

[0042] 1. Sample collection:

[0043] Collect 5-7.5ml of peripheral blood from breast cancer patients, anticoagulate with EDTA, and use it within 4 hours (or use it within 48 hours after storage at 4 degrees).

[0044] 2. Select magnetic beads:

[0045] 2.1 Processing of magnetic beads:

[0046] Mix the magnetic beads evenly, use a pipette to blow gently, and do not use a vortexer;

[0047] Use PBS buffer solution to wash, and do not touch the microspheres when washing;

[0048] Draw more magnetic beads than the required sample volume and add them to a 1.5ml centrifuge tube;

[0049] Put it on the magnet for 1 minute, discard the supernatant;

[0050] Wash 3 times with 1ml PBS to remove preservatives;

[0051] Remove the supernatant;

[0052] · Aspirate 100ul microspheres containing PBS with the same volume as the original one.

[0053] 2.2 Selection of tumor cells

[0054] Put 5ml of peripheral blood into a ...

Embodiment 3

[0073] Example 3 Detection of Breast Cancer Cell Tumor Markers

[0074] 3.1 Kit preparation

[0075] Keep the test tube at room temperature;

[0076] Put the RNase-free water in the kit at room temperature;

[0077] Place the centrifuge tube above the tube containing the supernatant on ice;

[0078] · Prepare magnetic beads.

[0079] 3.2 Preparation of oligonucleotides

[0080] 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);

[0081] · Transfer to a 1.5ml tube;

[0082] • Wash twice with lysis / binding buffer.

[0083] 3.3 Binding mRNA to magnetic beads

[0084] Add 20 μl of magnetic beads to each cell lysis sample;

[0085] • Incubate for 10 minutes.

[0086] 3.4 Purification of mRNA

[0087] Wash twice with buffer solution;

[0088] Wash the magnetic beads twice with buffer;

[0089] Wash again with 100 μl Tris-HCl buffer;

[0090] • Suspend the magneti...

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 breast cancer cells (malignant tumor cells) from the circulating blood of breast cancer patients, especially capable of separating A detection kit for extracting and identifying free breast cancer cells remaining in the patient's blood after radiotherapy and chemotherapy. Background technique

[0002] As the killer of malignant tumors, breast cancer has an obvious rising trend. The high incidence of breast cancer is increasingly harmful to human beings, especially women. For the early diagnosis and screening of breast cancer and the prognosis of treatment effect, as well as the evaluation and prediction of the curative effect after chemotherapy and radiotherapy, it is of great practical significance to reduce the mortality rate of breast cancer and increase the 5-year survival rate.

[0003] ...

Examples

Embodiment 1

[0038] The preparation of embodiment 1 magnetic beads

[0039] Preparation of antibody immunomagnetic beads: the antibodies used were anti-BerEP4 monoclonal antibody, anti-cytokeratin monoclonal antibody and anti-HER2 monoclonal antibody. In the present invention, the above three kinds of antibodies are labeled separately and then mixed for use. It is also possible to select one or two antibodies for separate labeling and mixed use.

[0040] 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

[0041] The separation of embodiment 2 breast cancer cells

[0042] 1. Sample collection:

[0043] Collect 5-7.5ml of peripheral blood from breast cancer patients, anticoagulate with EDTA, and use it within 4 hours (or use it within 48 hours after storage at 4 degrees).

[0044] 2. Select magnetic beads:

[0045] 2.1 Processing of magnetic beads:

[0046] Mix the magnetic beads evenly, use a pipette to blow gently, and do not use a vortexer;

[0047] Use PBS buffer solution to wash, and do not touch the microspheres when washing;

[0048] Draw more magnetic beads than the required sample volume and add them to a 1.5ml centrifuge tube;

[0049] Put it on the magnet for 1 minute, discard the supernatant;

[0050] Wash 3 times with 1ml PBS to remove preservatives;

[0051] Remove the supernatant;

[0052] · Aspirate 100ul microspheres containing PBS with the same volume as the original one.

[0053] 2.2 Selection of tumor cells

[0054] Put 5ml of peripheral blood into a ...

Embodiment 3

[0073] Example 3 Detection of Breast Cancer Cell Tumor Markers

[0074] 3.1 Kit preparation

[0075] Keep the test tube at room temperature;

[0076] Put the RNase-free water in the kit at room temperature;

[0077] Place the centrifuge tube above the tube containing the supernatant on ice;

[0078] · Prepare magnetic beads.

[0079] 3.2 Preparation of oligonucleotides

[0080] 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);

[0081] · Transfer to a 1.5ml tube;

[0082] • Wash twice with lysis / binding buffer.

[0083] 3.3 Binding mRNA to magnetic beads

[0084] Add 20 μl of magnetic beads to each cell lysis sample;

[0085] • Incubate for 10 minutes.

[0086] 3.4 Purification of mRNA

[0087] Wash twice with buffer solution;

[0088] Wash the magnetic beads twice with buffer;

[0089] Wash again with 100 μl Tris-HCl buffer;

[0090] • Suspend the magneti...

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