A method for real-time detection of the number of rare cells based on low-field nmr

A rare cell, real-time detection technology, applied in the field of molecular biology detection, can solve problems such as long centrifugation time, and achieve the effects of low price, simple and quick operation, and sensitive detection process.

Active Publication Date: 2017-02-08
上海美吉医学检验有限公司
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Problems solved by technology

The disadvantages of this method are: ① longer centrifugation time; ② need to prepare inert gradient medium solution

Method used

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  • A method for real-time detection of the number of rare cells based on low-field nmr
  • A method for real-time detection of the number of rare cells based on low-field nmr
  • A method for real-time detection of the number of rare cells based on low-field nmr

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] (1) Sample preparation

[0042] Preparation of samples to be tested

[0043] 1.1 Obtain an anticoagulated blood sample (~10 ml) from a breast cancer model mouse containing a mixed cell population suspected of containing circulating rare cells. Collected specimens should be processed the same day! Keeping time at room temperature away from light should not exceed 24 hours;

[0044] 1.2 Ficoll density gradient centrifugation method to obtain candidate mixed cell populations

[0045] 1) Add an appropriate amount of cell layering solution (prepared from Ficoll solution) to the bottom of sterile centrifuge tube A, then dilute the above anticoagulant test sample with PBS solution (2:1), and gently add On top of the layered liquid, the two form a clear interface.

[0046] 2) Centrifuge horizontally at 400×g for 30 minutes (the increase and decrease of the centrifuge speed should be uniform and stable to ensure the formation of a clear interface).

[0047] 3) After centrif...

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Abstract

The invention discloses a method for detecting the number of rare cells in real time based on low field NMR (nuclear magnetic resonance). The method comprises the following steps: (1) preparing a standard sample and a to-be-tested sample of target rare cell suspension; (2) respectively adding an NMR contrast media coupled with a target rare cell specific expression antibody, mixing uniformly, and immediately and respectively carrying out NMR relaxation time detection before immune incubation; (3) carrying out constant-temperature immune incubation on the standard sample and the to-be-tested sample, and carrying out NMR relaxation time detection at same interval; (4) drawing an immune incubation time-relaxation time value standard curve, an immune incubation time-relaxation time variable standard curve and an incubation cell number-relaxation time variable maximum curve of the standard sample; and (5) computing the relaxation time variable of the to-be-tested sample, and measuring the concentration of rare cells in the to-be-tested sample according to the curves obtained in the step (4). The method can be used for detecting the concentration and the number of rare cells simply, conveniently, quickly, highly sensitively and directly in real time and with high specificity.

Description

technical field [0001] The invention relates to molecular biology detection technology, in particular to a detection method for rare cells in biological fluid samples. Background technique [0002] Rare cells refer to some atypical cells in biological fluid samples (including blood, pleural effusion, ascites, urine, cerebrospinal fluid, etc.). has important guiding significance. Therefore, finding an accurate and rapid detection method for rare cells will become an urgent problem to be solved. However, the concentration of rare cells in biological fluids is very low, with a ratio of about 1:10 to non-target cells 7 , cannot be counted with traditional techniques, so a simple, accurate and fast method is urgently needed to solve this difficult problem. [0003] Since the content of rare cells in body fluids is very small, effective enrichment steps are required to identify and identify them in subsequent experiments. At present, the methods widely used in the detection an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N24/08
Inventor 张祥林
Owner 上海美吉医学检验有限公司
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