POCT detection device based on thermal convection fluorescence PCR amplification

ABSTRACT

This article is based on the principle of thermal convection PCR and nucleic acid fluorescence intensity detection technology. The principle of thermal convection PCR is to form a temperature difference by separately controlling the upper temperature and the bottom temperature of the reaction tube. The lower temperature liquid at the upper part has relatively high density or specific gravity, and the upper and lower liquids will produce convection, which drives the flow of molecules in the tubular chamber. The reaction solution is formed into thermal convection in the reaction test tube and subjected to different temperatures, so as to meet the required conditions for the reaction of different enzymes, and realize the pre-denaturation, annealing and extension processes in the nucleic acid PCR amplification in a short time. Nucleic acid fluorescence intensity detection technology involves embedded system design for device control and signal analysis, optical system design for optical signal filtering and collection, and differential amplifier circuit design. The embedded system design is based on the development of precise temperature control system, motion system and signal analysis system based on Stm32 single-chip microcomputer. The temperature control system includes independent temperature control to control the heaters at the bottom of the reaction tube and the top of the reaction tube respectively;the motion system includes sample switching and switching of the light source in the imaging system. The optical system design includes 540nm FAM excitation light source, 570nm CY3 excitation light source and spherical lens focusing excitation system. This device uses a photodiode to convert the optical signal into an electrical signal, and then amplifies the collected electrical signal with a two-stage operational amplifier through a two-color light differential amplifier circuit, and then uses the signal analysis system to record and display the electrical signal changes in real time, and Make a qualitative analysis. This device not only has the advantages of low cost and high sensitivity, but also solves the key problem of the long time (more than 2 hours) of the whole process of real-time fluorescent quantitative PCR in the detection of new crown nucleic acid and cannot be screened quickly on site. The PCR time of this device is from 2 The hour is reduced to 30 minutes, which is suitable for POCT inspections, and achieves rapid screening goals for crowds of people, which is conducive to responding to acute nucleic acid detection and large-scale nucleic acid detection. This device is currently used with COVID-19 detection reagents to detect new coronaviruses, and realize the detection of 20 copies of nucleic acid sensitivity within 30 minutes. Four samples can be processed in batches at a time, and the sample size for single processing can be increased appropriately according to needs. This device provides rapid and sensitive screening methods for global epidemic prevention and control, and is of great significance to improve human health. This device can also be applied to other rapid nucleic acid detection fields. With different nucleic acid detection reagents, this device can detect different gene loci, and has a broad development space and application fields. © 2023 SPIE.