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Continuous volume gradient capillary digital PCR method and kit

A capillary, digital technology, applied in the field of digital PCR, can solve the problem of high cost, achieve the effect of cost saving, good application prospect and accurate detection method

Active Publication Date: 2019-02-15
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problems of high cost and the need for pre-experiment in the prior art, the present invention provides a continuous volume gradient capillary digital PCR method

Method used

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  • Continuous volume gradient capillary digital PCR method and kit

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Embodiment

[0029] Embodiment A kind of continuous volume gradient capillary digital PCR method

[0030] 1. Continuous Volume Gradient Capillary Preparation

[0031] Use a capillary with an inner diameter of 1mm to make a continuous volume gradient capillary. Under the grill of an alcohol lamp, pull the capillary evenly and forcefully until it breaks, and trim the tip of the capillary to expose the tip hole unobstructed. A capillary having a thick end inner diameter of 1 mm, a thin end inner diameter of 20 μm, and a length of 3 cm was obtained.

[0032] 2. Oil phase preparation

[0033] First prepare oil with a density close to the density of water. In this example, castor oil (0.98g / l) and paraffin oil are mixed evenly according to a volume ratio of 3:1 and cholesterol is added. The ratio is 100ml of castor oil and excess cholesterol is added to dissolve it to saturation. State, add 0.5% Span 80 or 0.1% tween 60 surfactant, shake evenly, make the mixture of castor oil, paraffin oil and...

experiment example

[0042] Experimental example Artificial EGFR gene quantitative test

[0043] In this experimental example, the artificially synthesized EGFR gene was used as the test template.

[0044] 1. Method

[0045] 1.1 Continuous Volume Gradient Capillary Preparation

[0046] Use a capillary with an inner diameter of 1mm to make a continuous volume gradient capillary. Under the grill of an alcohol lamp, pull the capillary evenly and forcefully until it breaks, and trim the tip of the capillary to expose the tip hole unobstructed. A capillary having a thick end inner diameter of 1 mm, a thin end inner diameter of 20 μm, and a length of 3 cm was obtained.

[0047] 1.2 Preparation of oil phase

[0048] First prepare oil with a density close to the density of water. In this example, castor oil (0.98g / l) and paraffin oil are mixed evenly according to a volume ratio of 3:1 and cholesterol is added. The ratio is 100ml of castor oil and excess cholesterol is added to dissolve it to saturation. ...

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Abstract

A continuous volume gradient capillary digital PCR method comprises the following steps: uniformly mixing a PCR reaction system containing fluorescent groups and an oil phase of which the density is equivalent to the density of water according to a volume ratio of 1 to 2-4; placing a mixture in a capillary with internal diameter continuously changed from large to small to perform PCR reaction; counting a microdroplet number and a microdroplet radius in an area with positive microdroplet ratio of 25-55% by using optical signal collection equipment; and calculating a PCR template copy number. The continuous volume gradient capillary digital PCR method provided by the invention has the advantages that the cost of digital PCR can be reduced and the operation process is simplified.

Description

technical field [0001] The invention relates to the field of digital PCR, in particular to a continuous volume gradient capillary digital PCR method. Background technique [0002] Modern biological research, especially medical research, often involves the need for quantitative analysis of nucleic acids. For example, the detection of certain free DNA and its content in blood can guide the clinical diagnosis of certain cancers and monitor the effect of cancer treatment. In the past, relative quantification was mainly performed by fluorescent quantitative PCR, that is, a transcript of a gene with stable expression was selected as a reference to judge the amount of the target nucleic acid. However, this method is susceptible to the interference of non-target nucleic acid molecules (background noise), and is only suitable for qualitative or low-precision detection, and cannot meet the detection of some target nucleic acid molecules with particularly rare content. [0003] Digit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2563/107C12Q2563/159
Inventor 李孝锦康焰
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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