Method and system for digital quantitative analysis of nucleic acid amplification based on micro-droplet

A technology for quantitative analysis and nucleic acid amplification reaction, which is applied in the field of quantitative analysis methods and systems for digital nucleic acid amplification based on microdroplets, and can solve problems such as high cost and unfavorable digital nucleic acid applications.

Active Publication Date: 2015-03-25
SICHUAN MACCURA BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above-mentioned methods rely on relatively complex fluid drive devices, etc., wh

Method used

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  • Method and system for digital quantitative analysis of nucleic acid amplification based on micro-droplet
  • Method and system for digital quantitative analysis of nucleic acid amplification based on micro-droplet
  • Method and system for digital quantitative analysis of nucleic acid amplification based on micro-droplet

Examples

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Embodiment 1

[0095] The vibrating device is an electromagnetic dotting timer powered by an AC power supply with a voltage of 6V and a frequency of 50Hz. After the power is turned on, the vibrating piece of the electromagnetic timer vibrates at a frequency of 50 times per second. The amplitude of the vibration is 4mm. The micropipe used is a pointed quartz capillary with a length of 5 cm, an inner diameter of 75 μm, and an outer diameter of 150 μm. The inner diameter of the tip is 30 μm and the outer diameter is 60 μm. One end of the quartz capillary was connected to a 50 μL micro-syringe (Shanghai Gaoge) filled with mineral oil through a Teflon tube with an inner diameter of 300 μm, and the connection between the capillary and the Teflon tube was sealed with epoxy resin. The microsyringe is fixed on a microsyringe pump (Harvard Instruments, USA), and a fixed volume sample is drawn or injected at a set flow rate. The reservoirs used are transparent polystyrene flat-bottomed 96-well plates...

Embodiment 2

[0104] According to the method of Example 1, a droplet with a volume of 4nL was obtained and a nucleic acid amplification reaction was performed; the difference was that the flow rate of the syringe pump was 200nL / s.

[0105] Generated droplets see Figure 6 , Figure 6 Microscopic view of a droplet generated for Example 2 of the present invention; droplet volume see image 3 . Droplets that produce a fluorescent signal see Figure 7 , Figure 7 It is a schematic diagram of the reaction of droplets with different volumes in Examples 2 to 4 of the present invention.

Embodiment 3

[0107] According to the method of Example 2, a droplet with a volume of 1.7 nL was obtained and a nucleic acid amplification reaction was performed; the difference was that the flow rate of the syringe pump was 80 nL / s, and the injection volume was 2.5 μL.

[0108] Generated droplets see Figure 8 , Figure 8 Microscopic view of a droplet generated for Example 3 of the present invention; droplet volume see image 3 . Droplets that produce a fluorescent signal see Figure 7 .

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Abstract

The invention provides a method and a system for digital quantitative analysis of nucleic acid amplification based on micro-droplet. The method comprises the following steps: preparing a to-be-detected nucleic acid amplification reaction liquid which includes a to-be-detected nucleic acid template, a reaction buffer water solution, deoxyribonucleoside triphosphate, a primer, polymerase and a product marking substance; loading the prepared to-be-detected nucleic acid amplification reaction liquid in a micro-pipeline of which the two ends are both provided with an opening, wherein the micro-pipeline is arranged above an open container, and the open container contains an oily liquid containing a surfactant; enabling the opening of one end of the micro-pipeline to do up-and-down reciprocating vibration on the surface of a liquid in the open container or do leftward-and-rightward reciprocating vibration below the liquid surface so as to generate a plurality of droplets which are flatly laid at the bottom of the open container; performing nucleic acid amplification reaction on the plurality of droplets in the open container; acquiring product signals generated after the nucleic acid amplification reaction is ended, and performing quantitative analysis on the nucleic acid template. The digital nucleic acid amplification analysis method can be used for implementing quantitative detection on low-concentration nucleic acid substances in a micro-system and is simple and convenient to operate, high in efficiency and low in cost.

Description

technical field [0001] The present application relates to the technical field of nucleic acid quantitative analysis, in particular to a micro-droplet-based digital nucleic acid amplification quantitative analysis method and system. Background technique [0002] The traditional quantitative PCR (Polymerase Chain Reaction, Polymerase Chain Reaction) technology is based on the exponential amplification of nucleic acid templates during the reaction process, and the quantification of nucleic acid templates can be achieved by comparing the number of amplification cycles and analyzing the amount of PCR products after amplification . However, there are many factors that cause the traditional quantitative PCR method to have certain limitations and inaccuracies. For example, the amplification is not exponential at the beginning, which will affect the accuracy of the analysis method; Difficult to detect or can only be detected if the amplification is present twice or more, etc. [00...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12M1/38
CPCC12Q1/6851C12Q2563/159
Inventor 杜文斌李昂徐鹏盛广济
Owner SICHUAN MACCURA BIOTECH CO LTD
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