High-throughput PCR micro-droplet fluorescent detection device and method

Abstract

The present invention discloses a high-throughput PCR micro-droplet fluorescent detection device including a micro-droplet chip, a sample charging system, a temperature control system and a fluorescent detection system. The micro-droplet chip includes a substrate layer and a closed cover plate layer, a reaction cavity is formed between the cover plate layer and the substrate layer, and the cover plate layer is provided with a sample charging hole; the sample charging system includes a micropin, a sample charging driving mechanism, a pressure driving mechanism and a sample charging box used foraccommodating samples and raw materials required for PCR, and the pressure driving mechanism provides pressure for the sample charging box so that the samples and the raw materials in the sample charging box are injected into the corresponding reaction cavity by the micropin; the temperature control system is used for detecting and providing a temperature for PCR circulation; and the fluorescentdetection system is used for obtaining excited fluorescence in the reaction cavity. By using the high-throughput PCR micro-droplet fluorescent detection device provided by the present invention, the micro-droplet preparation, nucleic acid amplification and detection of the samples are integrated by virtue of the integration and miniaturization characteristics of the micro-droplet chip, so that theoperation process is simplified, and external pollution and cross pollution of nucleic acids are effectively avoided.

Description

technical field [0001] The invention relates to the field of nucleic acid detection in the field of biomedical testing, in particular to a high-throughput PCR droplet fluorescence detection device and method. 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 to suspend the sample mixture into dropl...

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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 detection (more than 4);

[0008] (4) The transfer process of the two droplets will result in the loss of droplets and possible external pollution;

[0009] (5) The thermal inertia of the heating block of the conventional PCR amplification instrument is large, and the thermal cycle time is long (about 2 hours)

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