A microfluidic system for rapid nucleic acid analysis based on real-time convective PCR at point-of-care testing

Abstract

A microfluidic system for rapid nucleic acid analysis based on real-time convective PCR is developed. To perform ‘sample-in, answer-out’ nucleic acid analysis, a microfluidic chip is developed to efficiently extract nucleic acid, and meanwhile convective PCR (CPCR) is applied for rapid nucleic acid amplification. With an integrated microfluidic chip consisting of reagent pre-storage chambers, a lysis & wash chamber, an elution chamber and a waste chamber, nucleic acid extraction based on magnetic beads can be automatically performed for a large size of test sample within a limited time. Based on an easy-to-operate strategy, different pre-stored reagents can be conveniently released for consecutive reaction at different steps. To achieve efficient mixing, a portable companion device is developed to introduce properly controlled 3-D actuation to magnetic beads in nucleic acid extraction. In CPCR amplification, PCR reagent can be spontaneously and repeatedly circulated between hot and cool zones of the reactor for space-domain thermal cycling based on pseudo-isothermal heating. A handheld real-time CPCR device is developed to perform nucleic acid amplification and in-situ detection. To extend the detection throughput, multiple handheld real-time CPCR devices can be grouped together by a common control system. It is demonstrated that influenza A (H1N1) viruses with the reasonable concentration down to 1.0 TCID 50 /ml can be successfully detected with the microfluidic system.

Advances in Simple, Rapid, and Contamination-Free Instantaneous Nucleic Acid Devices for Pathogen Detection

“…The principle of how device-convective PCR works is shown in Figure 2C. A microfluidic chip for automated nucleic acid extraction and a handheld real-time CPCR system for rapid amplification were developed by Xu et al [130]. The amplification time has been significantly reduced to 30 min using CPCR compared to the conventional PCR.…”

Sophisticated and precise: design and implementation of a real-time optical detection system for ultra-fast PCR

“…The absorbance versus concentration deviates from linearity in highly concentrated solutions due to the limitation of the linear range of the Lambert-Beer law. [24][25][26] During uorescence detection system performance testing, gradient stability and linearity are oen screened, and such deviations can introduce a certain amount of error during nal instrument performance verication. To this end, we conducted tests based on the FAM channel, studied the spatial intensity distribution of uorescence emission over a wide concentration range, obtained the relationship between the received uorescence intensity and the concentration of uorescent substances, and established a uorescence model that includes the spatial attenuation effect to avoid similar spatial attenuation problems in subsequent experiments and reduce misjudgment of the instrument performance.…”

On-Chip Nucleic Acid Purification Followed by ddPCR for SARS-CoV-2 Detection

“…Generally, nucleic acid purification is required before PCR to remove inhibitors prior to the amplification reaction [3]. Researchers have also tried to introduce purification-free methods to simplify the diagnosis procedures.…”