Large-area digital PCR microsphere fluorescence high-flux detection device and method

A detection device and fluorescence detection technology, applied in the field of biomedical testing, can solve the problems of inability to real-time qPCR, long thermal cycle time, and difficulty in realizing multiple digital PCR detection, so as to improve detection accuracy and time limit, improve medical detection technology, improve The effect of clinical testing methods

Pending Publication Date: 2017-12-15
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] (1) The flow detection speed is slow, the detection speed is 200-500 pieces/s, the detection process takes a long time, and the throughput is low;
[0006] (2) End-point detection can only be performed on a single droplet, and real-time qPCR is not possible, which reduces specificity and sensitivity;
[0007] (3) The optical path hardware is complicated, and it is difficult to realize multiple digital PCR dete

Method used

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  • Large-area digital PCR microsphere fluorescence high-flux detection device and method
  • Large-area digital PCR microsphere fluorescence high-flux detection device and method
  • Large-area digital PCR microsphere fluorescence high-flux detection device and method

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example 1

[0168] The parameters of the digital PCR microfluidic chip to be detected are: to achieve double digital PCR, use the first dye and the second dye, the excitation wavelengths are 488nm and 532nm, the fluorescence wavelengths are 515nm and 560nm, respectively, and the droplet digital PCR microfluidic The control chip integrates 48 single-sample detection structures, and the overall chip is 150mm long and 100mm wide.

[0169] see Figure 17 , for this chip, a real-time fluorescence image acquisition device for single-layer digital PCR droplets is proposed, including:

[0170] Objective lens device 200, including two objective lenses 2011, 2012 with magnifications of 4X and 10X;

[0171] The light source device 300 is used for fluorescence excitation of digital PCR microdroplets, including two lasers 3011, 3012, two sets of shutters 3081, 3082, two sets of lenses 3021, 3022, and a dichromatic mirror 3091; wherein, the wavelength of the laser 3011 is 488nm , the laser 3012 has a...

example 2

[0190] The parameters of the digital PCR microfluidic chip to be detected are: six-fold digital PCR, and the dyes used are:

[0191] Fluorescent dye 1: excitation wavelength 390nm, fluorescence wavelength 420nm;

[0192] Fluorescent dye 2: excitation wavelength 475nm, fluorescence wavelength 522nm;

[0193] Fluorescent dye 3: excitation wavelength 525nm, fluorescence wavelength 577nm;

[0194] Fluorescent dye 4: excitation wavelength 572nm, fluorescence wavelength 628nm;

[0195] Fluorescent dye 5: excitation wavelength 630nm, fluorescence wavelength 675nm;

[0196] Fluorescent dye 6: excitation wavelength 650nm, fluorescence wavelength 710nm;

[0197] The droplet digital PCR microfluidic chip integrates 48 single-sample detection structures, and the overall chip is 150mm long and 100mm wide. For this chip, see Figure 18 , a device for real-time fluorescence image acquisition of digital PCR droplets for monolayer tiling is proposed, including:

[0198] Objective lens de...

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Abstract

The invention discloses a large-area digital PCR microsphere fluorescence high-flux detection device, which comprises a fluorescence exciting light path and a fluorescence detection light path, wherein along the fluorescence exciting light path, exciting light emitted by a light source device sequentially passes through a focusing device and an objective lens device to be emitted onto a digital PCR chip of an objective table; along the fluorescence detection light path, the fluorescence generated by the digital PCR chip being excited by the exciting light sequentially passes through the objective lens device, the focusing device and an imaging device to be imaged and is then transmitted to an image collection sensor; the objective table is also provided with a temperature control device; the temperature control device controls the circulation temperature of the reaction of the digital PCR chip in real time. The invention discloses a large-area digital PCR microsphere fluorescence high-flux detection method. The higher detection efficiency and the large-area high-flux detection accuracy can be obtained. The method and the device can realize the real-time digital qPCR detection with high flux, high sensitivity and high specificity; the important significance is realized on improving the performance, the precision and the flux of a digital PCR detection instrument.

Description

technical field [0001] The invention relates to the field of nucleic acid detection in the field of biomedical testing, in particular to a device and method capable of realizing large-area digital PCR droplet fluorescence high-throughput detection. Background technique [0002] In fields such as early diagnosis and screening of tumors, blood virus typing and load analysis, and noninvasive prenatal diagnosis, high-speed, high-sensitivity, and high-specificity detection of ultra-low-concentration nucleic acids is required, and conventional PCR is increasingly unable to meet the requirements. need. In order to achieve this function, people have developed a digital PCR technology on the basis of PCR, which improves the detection sensitivity by 1 to 2 orders of magnitude. [0003] The "divide and conquer" detection strategy adopted by droplet digital PCR uses the sample and PCR reagent mixture as the dispersed phase, oil as the continuous phase, and uses microfluidic technology ...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6402G01N21/6486
Inventor 周武平黎海文蒋克明刘聪张涛印晨宇
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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