Detection method of nucleic acid in micro sample

A detection method and sample technology, applied in the field of nucleic acid detection in trace samples, can solve the problems of complex operation and low specificity in the separation and detection process of biological components, reduce the difficulty of sampling, reduce requirements, and realize high-throughput gene The effect of detection

Pending Publication Date: 2017-05-10
上海默里科基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] An object of the present invention is to solve the problems of complex operation and low specificity in the separation and detection process of biological components in the prior art

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. Isolation of leukocytes and detection of nucleic acid from human whole blood

[0042] (1) coupling the CD45 specific antibody to the shell of the capture complex;

[0043] (2) Take 3 microliters of human fingertip blood samples and place them in a container. At 37 degrees Celsius, add 100 microliters of HEPES buffer (pH7.4), add the capture complex, shake, and the capture complex specifically binds to leukocytes to form a capture complex. Body-leukocyte conjugates, use a magnet to absorb the capture complex on the outer wall of the container to the inner wall of the container, remove waste liquid, add rinsing solution to wash the capture complex-leukocyte conjugate, and remove waste liquid;

[0044] (3) Add the reagents required for PCR (including buffer, Taq polymerase, dNTPs, primers, water, etc.) into the capture complex-leukocyte conjugate, and directly enter the fluorescent quantitative PCR program. Leukocytes will be lysed and released nucleic acid as ...

Embodiment 2

[0045] Example 2. Isolation of mouse thymus epithelial cells and detection of nucleic acid from thymus tissue culture fluid

[0046] (1) coupling the mAb RE-4D8 antibody to the shell of the capture complex;

[0047] (2) Take 3 microliters of thymus tissue culture fluid samples and mix them with the capture complex, place them in a container, add 100 microliters of Tris-EDTA buffer (pH7.6), and stir the mixture gently with a vortex mixer for 15 seconds , let stand at 37 degrees Celsius for 3 minutes, the capture complex specifically binds to mouse thymus epithelial cells to form a capture complex-epithelial cell conjugate, use a magnet to absorb the capture complex on the outer wall of the container to the inner wall of the container, remove the waste liquid, add PBS buffer (pH 7.4) washes the capture complex-epithelial cell combination to remove waste liquid;

[0048] (3) Add reagents required for PCR (including buffer, Taq polymerase, dNTPs, primers, water, etc.) into the ca...

Embodiment 3

[0050] Example 3. Isolation of M13 phage and detection of nucleic acid from bacterial culture fluid

[0051] (1) coupling the anti-M13 antibody to the shell of the capture complex;

[0052] (2) Take 100 microliters of bacterial culture fluid samples and place them in a container, dilute the antigen to 10 μg / ml with Hanks solution, add the capture complex to mix, and stir the mixture gently with a vortex mixer for 15 seconds, and place it at 37 degrees Celsius Incubate for 3 minutes, the capture complex specifically binds to the M13 phage to form a capture complex-phage conjugate, use a magnet to absorb the capture complex on the outer wall of the container to the inner wall of the container, remove the waste liquid, add PBS buffer (pH7.4) for The capture complex-phage conjugate is washed to remove waste liquid;

[0053] (3) Add the reagents required for PCR (including buffer, Taq polymerase, dNTPs, primers, water, etc.) to the capture complex-phage conjugate, and directly ent...

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PUM

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Abstract

The invention relates to a detection method of nucleic acid in a micro sample. The detection method includes acquiring a composite capture body comprising a magnetic core and a shell covering the magnetic core, wherein the shell is coupled with an antibody which has specific affinity to specific epitope of cells or viruses in the sample; putting the sample in a container, and contacting the sample with the composite capture body to enable the composite capture body to combined with the cells or viruses; applying a magnetic field to the container to adsorb the composite capture body to the inner wall of the container, and removing waste liquid; adding rinsing liquid to wash the composite capture body to obtain a composite capture body-cell combination or composite capture body-virus combination, and removing waste liquid; amplifying and detecting the composite capture body-cell combination or composite capture body-virus combination. By the detection method, the problems of complexity in operation of biological element separation and detection and low specificity in the prior art are solved, and automation and high-throughput treatment of separation and detection processes are realized easily.

Description

technical field [0001] The invention relates to the field of nucleic acid detection, in particular to a method for detecting nucleic acid in trace samples. Background technique [0002] Since the advent of PCR technology in the 1980s, its application in molecular biology-related fields has become more and more extensive. Especially in recent years, with the improvement of people's requirements for the accuracy and sensitivity of clinical diagnosis, and the introduction and promotion of the concept of individualized medicine, PCR technology has been rapidly and widely used in clinical diagnosis due to its advantages of rapidity, sensitivity and specificity in clinical diagnosis. Diagnosis and treatment, by detecting genes in patient samples to help doctors determine medication and treatment options. [0003] The extraction of sample nucleic acid is the prerequisite for the application of PCR technology. Human peripheral blood plays an important role in the immune response an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12M1/42C12M1/34
CPCC12Q1/6804C12Q2563/143
Inventor 孙相鑫魏宏泉李兴旺万戈江王晓维魏然
Owner 上海默里科基因科技有限公司
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