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Mammaglobin mRNA detection method and reagent thereof

A detection method and a technology of a detection kit, which are applied in DNA/RNA fragments, recombinant DNA technology, microbial determination/inspection, etc., can solve the problems of high environmental purity requirements, high false positive rate of antibody specificity requirements, etc.

Inactive Publication Date: 2013-10-16
SUZHOU YOULIN BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The first problem to be solved by the present invention is to overcome the problems that the existing specific detection of human mammaglobulin requires high environmental purity, very high antibody specificity and high false positive rate, and provides a micro-mammary gland in circulating blood A specific detection method for cancer cells. This method is easy to use, sensitive, strong specificity, good repeatability, quick results, no need for complicated instruments and special skills, and can be directly detected by using unpurified samples without precision. Mutual interference between aptamers and target molecules (MAM mRNA) and other non-specific nucleic acid sequences

Method used

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  • Mammaglobin mRNA detection method and reagent thereof
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  • Mammaglobin mRNA detection method and reagent thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0069] Embodiment 1 detection uses the preparation of MAM mRNA sample and its contrast

[0070] 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 (pH7.2), and each dilution solution contained equal volumes from the equivalent of 10 5 (Group A), 10 4 (Group B), 10 3 (Group C), 10 2 (Group D) and 10 1 (Group E) total RNA amount of MDA-MB361 breast cancer cells.

[0071] According to the above method, the tot...

Embodiment 2

[0072] Synthesis and preparation of embodiment 2 aptamers

[0073] Take 12 microliters of magnetic body solution with carboxy-terminus in a 1.5ml centrifuge tube, wash with 0.1M imidazole buffer three times (each time add 150 microliters of 0.1M imidazole buffer (pH7.0) and mix well, then centrifuge. Discard the supernatant), add 300 microliters of 0.1M imidazole buffer (pH7.0) containing 0.03M EDC, shake the centrifuge tube slowly for 20 minutes, and then add 12pmol 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 100 mM NaCl, pH 7.2) and blot dry) for three times. Unbound free first aptamer was removed, and then dissolved by adding 240 microliters of dissolving soluti...

Embodiment 3

[0075] Example 3 Determination of the concentration of the first aptamer and the second aptamer

[0076] In a series of reaction tubes, the total RNA (1 microgram) of MDA-MB361 cells known to contain the target molecule (MAM mRNA) was added, and the first aptamer (2- 20 microliters, 1-10pmol) mixed, added PBS (0.1M, pH7.2) to 100 microliters, incubated at 37°C for 60 minutes; then placed the test tube on a magnetic separator to apply magnetic force, blotted the supernatant and used The washing solution was washed three times (300 microliters of washing solution (PBS solution containing 100 mM NaCl, pH 7.2) was added each time and blotted dry), 95 microliters of PBS buffer solution was added, mixed evenly, and then 5 microliters were added according to Example 2. The prepared second aptamer coupled with gold nanoparticles 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, and wa...

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Abstract

The invention provides a method for detecting and / or quantifying mammaglobin mRNA (messenger ribonucleic acid) in a sample. The method comprises the steps of providing first aptamers coupled with magnetic particles and second aptamers coupled with gold nanoparticles, mixing, preforming magnetic separation and detecting products of magnetic separation. The invention further relates to a to-be-detected blood sample pretreatment reagent, an intermediate product, a detection kit and the like, which are used in the method.

Description

technical field [0001] The invention relates to the technical field of nucleic acid detection methods, in particular, the invention relates to a specific detection method for detecting, identifying and / or quantifying a small amount of breast cancer cell globulin mRNA in human circulating blood. In addition, the present invention also relates to kits for the method, intermediate products and applications thereof. Background technique [0002] Breast cancer is one of the most common lethal cancers in women. Surgical resection is the first choice in the comprehensive treatment of breast cancer, but the recurrence rate and metastasis rate after breast cancer resection are high: cancer cells are easy to enter the peripheral blood from the primary tumor to generate Tumor metastasis is the main cause of breast cancer treatment failure. A large number of experiments have confirmed that the detection of Circulating Tumor Cells (CTCs) in circulating blood will be helpful for early di...

Claims

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Application Information

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