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Microdroplet digital PCR biochip

A biochip, droplet technology, applied in enzymology/microbiology devices, biomass post-processing, biomass pretreatment, etc., can solve the problems of high requirements and complex systems for fluid control systems, and achieves reduction of operation steps, High-throughput, fast effects

Inactive Publication Date: 2017-05-31
PILOT GENE TECH HANGZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The overall equipment system of these methods is relatively complicated, and the requirements for the fluid control system are relatively high.

Method used

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  • Microdroplet digital PCR biochip
  • Microdroplet digital PCR biochip
  • Microdroplet digital PCR biochip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Such as Figure 1~5 As shown, a droplet type digital PCR biochip includes a chip body formed by laminating an upper piece 1 and a lower piece 2, and the inside of the chip body is provided with a sampling chamber 3 and a droplet storage chamber on the upper piece 1. 4 and oil discharge chamber 5.

[0030] The droplet storage chamber 4 is a U-shaped structure, and the sample injection chamber 3 is a linear structure, which is arranged between the two arms of the U-shaped structure, and a plurality of droplet forming channels 6 arranged in parallel are arranged between the two arms; the oil discharge The cavity 5 is provided on the outer periphery of the U-shaped structure. A plurality of oil discharge holes 7 are provided between the droplet storage chamber 4 and the oil discharge chamber 5 . The heights of the droplet forming channel 6 and the oil discharge channel 7 are both smaller than the height of the droplet storage chamber 4, so that the connection between the dr...

Embodiment 2

[0039] The chip body is provided with a plurality of reaction zones, and each reaction zone is provided with a sampling chamber 3, a droplet storage chamber 4, and an oil discharge chamber 5, and the surface of the chip body is provided with the sampling chamber 3 and the drain chamber respectively. A sampling hole 8 and an oil discharge hole 9 connected to the oil chamber 5, a plurality of droplet forming channels 6 are provided between the sampling chamber 3 and the droplet storage chamber 4, and between the droplet storage chamber 4 and the oil discharge chamber 5 and the oil discharge channel 7 , the heights of the droplet forming channel 6 and the oil discharge channel 7 are all smaller than the height of the droplet storage chamber 4 . All the other structures are with embodiment 1.

Embodiment 3

[0041] Use the droplet type digital PCR biochip in embodiment 1, use 70% mineral oil+30% n-tetradecane+3%EM90+3%TritonX-100 as the oil phase composition (ratio of mineral oil and n-tetradecane Refers to the mass ratio of the main component, and the ratio of EM90 and TritonX-100 is the ratio of their respective mass to the main component.) The droplet preparation experiment was carried out, and PCR amplification was carried out. The specific process is as follows:

[0042] (1) Prepare the oil phase.

[0043] (2) Prepare the aqueous phase, that is, the PCR reaction system.

[0044] The template comes from non-small cell lung cancer (NSCLC) cell line H1975, which has two mutations, T790M and L858R.

[0045] The primer sequences are:

[0046] F: 5'-GCCTGCTGGGCATCTG-3';

[0047] R: 5'-TCTTTGTGTTCCCGGACATAGAC-3'

[0048] The probe sequence is:

[0049] 5'-FAM-ATGAGCTGCATGATGAG-MGB-NFQ-3', wherein FAM is a fluorescent reporter group, and NFQ is a fluorescent quencher group.

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Abstract

The invention discloses a microdroplet digital PCR biochip which comprises a chip main body, the chip main body is formed by fitting of an upper piece and a lower piece, the chip main body is internally provided with a sample feeding chamber, a droplet storage chamber and an oil discharge chamber, the surface of the chip main body is provided with a sampling hole and an oil discharge hole which are respectively communicated with the sample feeding chamber and the oil discharge chamber, a plurality of droplet formation channels and oil discharge channels are respectively arranged between the sample feeding chamber and the droplet storage chamber and the droplet storage chamber and the oil discharge chamber, and the height of the droplet formation channels and the height of the oil discharge channels are lower than the height of the droplet storage chamber. When a water phase enters the relatively-broader droplet storage chamber via the relatively-narrow droplet formation channels, when the water phase partly enters the droplet storage chamber, due to surface tension, the flow of the water phase is accelerated, the water phase in the droplet storage chamber is fractured to form droplets. The prepared droplets have uniform size, high preparation speed and high flux.

Description

technical field [0001] The invention relates to the technical field of biochip preparation, in particular to a droplet type digital PCR biochip. Background technique [0002] Fluorescence Quantitative Polymerase Chain Reaction (qPCR) has developed into a key routine technology in the field of molecular biology, which has greatly promoted the development of various fields of life science. However, there are many factors affecting the amplification efficiency during the PCR amplification process, and it is difficult to ensure that the amplification efficiency of the actual sample and the standard sample and between different samples are the same, which leads to the basis of its quantitative analysis-the cycle threshold (Ct value) is not constant. Therefore, the quantification of qPCR is only relative quantification, and its accuracy and reproducibility still cannot meet the requirements of quantitative analysis in molecular biology. In addition, due to the inhibitory effect ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/38C12M1/00
CPCB01J2219/00317B01L3/5027B01L7/52
Inventor 赵然聂梦月罗海贝
Owner PILOT GENE TECH HANGZHOU CO LTD
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