ABSTRACT
Nucleic acid detection (NAD) based on real-time polymerase chain reaction (real-time PCR) is gold standard for infectious disease detection. Magnetic nanoparticles (MNPs) are widely used for nucleic acid extraction (NAE) because of their excellent properties. Microfluidic technology makes automated NAD possible. However, most of the NAD microfluidic chips are too complex to be applied to point-of-care (POC) testing. In this paper, a simple-structure cartridge was developed for POC detection of infectious diseases. This self-contained cartridge can be divided into a magnetic-controlled NAE part, a valve-piston combined fluidic control part and a PCR chip, which is able to extract nucleic acid from up to 500 μL of liquid samples by MNPs and finish the detection process from "sample in” to "answer out” automatically. Performance tests of the cartridges show that it met the demands of automated NAD. Results of on-cartridge detection of hepatitis B virus (HBV) demonstrated that this system has good uniformity and no cross-contamination between different cartridges, and the limit of detection (LOD) of this system for HBV in serum is 50 IU/mL. Multiplex detections of severe acute respiratory syndrome coronaviruses 2 (SARS-CoV-2) with a concentration of 500 copies/mL were carried out on the system and 100% positive detection rate was achieved.
ABSTRACT
A portable nucleic acid detection (PNAD) system based on real-time polymerase chain reaction (real-time PCR) has been developed for point-of-care testing (POCT) of infectious disease pathogens. In order to achieve "sample-in, result-out" while keeping the system compact, the hardware system integrates optical, thermal and motion control modules in a limited space for nucleic acid extraction, purification, amplification and detection. Among these hardware modules, the fluorescence module is one of the most important modules, because its performance directly affects the accuracy and sensitivity of the testing results. In this paper, a miniaturized, high-sensitivity and integrated dual-channel fluorescence module have been proposed for the homemade PNAD system. Based on the principle of confocal optical path, two group of excitation-emission optical paths of different wavelengths are integrated in a small space. In terms of circuitry, a current-light dual negative feedback light emitting diode (LED) drive circuit is applied to improve the stability of the excited light source. All optical and electronic components are integrated in a metal box of 55 mm × 45 mm × 15 mm, that helps miniaturize the detection system. Two different modules have been assembled to fit various fluorescent dyes or probes with the set of excitation and emission as follow: module 1#: 470 nm/525 nm, 570 nm/630 nm; module 2#: 520 nm/570 nm, 630 nm/690 nm. Finally, hepatitis B virus (HBV) concentration gradient detection and multiplex detection of different gene targets of SARS-CoV-2 are carried out on the PNAD system equipped with these two fluorescence modules for evaluating their performances. Compared with the commercial real-time PCR instrument, our fluorescence module has good stability and detection sensitivity.