Specific detection method and kit for trace amount of breast cancer cells in circulating blood

A breast cancer cell and detection method technology, which is applied in the field of specific detection and kits for trace breast cancer cells in circulating blood, can solve the problems of high environmental purity requirements and high false positive rate of antibody specificity requirements

Active Publication Date: 2011-12-28
SUZHOU YOULIN BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The first problem to be solved by the present invention is to overcome the high requirement for environmental purity, very high requirement for antibody specificity and fake In order to solve the problem of high positive rate, a specific detection method for trace breast cancer cells in circulating blood is provided. It can be directly detected by using the sample to be tested without precision purification, and there will be no mutual interference between the aptamer and the target molecule (MAM mRNA) and other non-specific nucleic acid sequences

Method used

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  • Specific detection method and kit for trace amount of breast cancer cells in circulating blood
  • Specific detection method and kit for trace amount of breast cancer cells in circulating blood
  • Specific detection method and kit for trace amount of breast cancer cells in circulating blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Preparation of MAM mRNA samples and their controls for detection

[0077] MDA-MB361 cells were cultured in vitro, the adherent cells were digested with trypsin to suspend the cells, the culture medium was removed by centrifugation, and then normal saline was added to prepare a single cell suspension, and a certain amount of cells (10 7 )conduct experiment. According to the manufacturer's instructions, total cellular RNA (positive for MAM mRNA expression) was extracted from MDA-MB361 cells with the total cellular RNA extraction reagent, and the optical density (OD value) at 260 and 280 nm was measured to quantify the amount of total RNA. Then the total RNA was serially diluted with TE buffer (pH 7.2), and equal volumes of solutions of each dilution contained 5 (Group A), 10 4 (Group B), 10 3 (Group C), 10 2 (Group D) and 10 1 (Panel E) The amount of total RNA in MDA-MB361 breast cancer cells.

[0078] According to the above m...

Embodiment 2

[0080] Example 2 Synthesis and preparation of aptamers

[0081] Take 12 microliters of the carboxy-terminal magnetic body solution and place it in a 1.5ml centrifuge tube, wash it three times with 0.1M imidazole buffer (each time add 150 microliters of 0.1M imidazole buffer (pH7.0) and mix well, then centrifuge. Discard the supernatant), add 300 μl of 0.1M imidazole buffer (pH7.0) containing 0.03M EDC, shake the centrifuge tube slowly for 20 minutes, and then add 12 pmol of the first aptamer (5'-aaggaagccgctgtc-3 ') was added, mixed and incubated at 37°C for 1 hour, during which the centrifuge tube was continuously shaken slowly. Place the centrifuge tube on a magnetic separator to apply magnetic force, blot the supernatant and wash it three times with washing solution (add 300 μl of washing solution (PBS solution containing 100mM NaCl, pH 7.2) and blot dry) for three times. Unbound free first aptamer was removed, and then dissolved by adding 240 μl of dissolving solution...

Embodiment 3

[0084] Example 3 Determination of the concentration of the first aptamer and the second aptamer

[0085] In a series of reaction tubes, total RNA (1 μg) of MDA-MB361 cells known to contain the target molecule (MAM mRNA) was added, together with the first aptamer coupled to magnetic particles prepared according to Example 2 (2- 20 μl, 1-10 pmol), add PBS (0.1M, pH7.2) to 100 μl, incubate at 37°C for 60 minutes; then place the test tube on a magnetic separator to apply magnetic force, and blot the supernatant solution and washed three times with washing solution (300 μl of washing solution (PBS solution containing 100mM NaCl, pH 7.2) was added each time and blotted dry), adding 95 μl of PBS buffer, mixing, and then adding 5 μl according to the implementation The second aptamer coupled with gold nanoparticles prepared in Example 2 was incubated at 37°C for 60 minutes, and then the test tube was placed on a magnetic separator to apply magnetic force, the supernatant was blotted...

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Abstract

The invention provides a specific detection method for a trace amount of breast adenocarcinoma cells in circulatory blood volume, which comprises: mixing a first adaptor coupled with a magnetic particle and a second adaptor coupled with nano gold particles with a sample to be mixed separately or at the same time; and detecting, identifying and / or quantifying target molecules. In addition, the invention also relates to a reagent for treating the blood sample to be treated, an intermediate, a detection kit and the like, which are used in the method. The method is simple and convenient for use and has high sensitivity, high specificity, high repeatability, quick result delivery, no need of complex instruments, and no special skills. Particularly, even if the sample to be detected is detecteddirectly without accurate purification, the method can still keep practical sensitivity and specificity; and thus, the detection operation is facilitated greatly, the consumption of a reagent is saved, the detection process is accelerated, and the method and the kit can be promoted for actual industrial application.

Description

[0001] Technical field [0002] The invention involves the technical field of nucleic acid detection methods. Specifically, the invention involves the specific detection method of detection, identification and / or quantitative human circulation blood in the blood.In addition, the present invention also involves the kit, intermediate product and its application for this method. Background technique [0003] Breast cancer is one of the most common dead cancer in women. Surgical resection is the first choice in comprehensive breast cancer treatment.Tumor metastasis, and tumor metastasis is the main reason for the failure of breast cancer treatment.A large number of experiments have confirmed that the detection of tumor cells (CTC) in cycle blood will help early diagnosis of breast cancer, recurrence and metastasis monitoring, judging patient prognosis, and postoperative assistive treatment. [0004] Human breast globulbin (MAM) is a kind of breast tissue -specific protein logo found ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/115
Inventor 李为游绍进
Owner SUZHOU YOULIN BIO TECH
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