A sample-in-digital-answer-out system for rapid detection and quantitation of infectious pathogens in bodily fluids

Abstract

A variety of automated sample-in-answer-out systems for in vitro molecular diagnostics have been presented and even commercialized. Although efficient in operation, they are incapable of quantifying targets, since quantitation based on analog analytical methods (via standard curve analysis) is complex, expensive, and challenging. To address this issue, herein, we describe an integrated sample-in-digital-answer-out (SIDAO) diagnostic system incorporating DNA extraction and digital recombinase polymerase amplification, which enables rapid and quantitative nucleic acid analysis from bodily fluids within a disposable cartridge. Inside the cartridge, reagents are pre-stored in sterilized tubes, with an automated pipetting module allowing facile liquid transfer. For digital analysis, we fabricate a simple, single-layer polydimethylsiloxane microfluidic device and develop a novel and simple sample compartmentalization strategy. Sample solution is partitioned into an array of 40,044 fL-volume microwells by sealing the microfluidic device through the application of mechanical pressure. The entire analysis is performed in a portable, fully automated instrument. We evaluate the quantitative capabilities of the system by analyzing Mycobacterium tuberculosis genomic DNA from both spiked saliva and serum samples, and demonstrate excellent analytical accuracy and specificity. This SIDAO system provides a promising diagnostic platform for quantitative nucleic acid testing at the point-of-care. Graphical abstract ᅟ.

The vision of point-of-care PCR tests for the COVID-19 pandemic and beyond

“…(C) ID NOW, made by Abbott Laboratories, originally sought to diagnose seasonal flu; it has currently been converted into a COVID-19 system, based on an isothermal DNA amplification originally developed for the seasonal flu test [ 91 ]. (D) Bosch's new microfluidic system is capable of virus diagnostics; the sample preparation is integrated with a multiplexed end-point PCR [ 92 ]. …”

“…4 D), a molecular diagnostic device based on a microwell array that is capable of detecting the coronavirus in less than 2.5 h, using a fully sealed cartridge. The Vivalytic system performs sample preparation, after which it performs a multiplex PCR, followed by microarray-detection, to allow the identification of SARS-CoV-2 [ 92 ].…”

Accurate, rapid and low-cost diagnosis of Mycoplasma pneumoniae via fast narrow-thermal-cycling denaturation bubble-mediated strand exchange amplification

“…Therefore, these two traditional methods cannot meet the requirement for accurate, rapid and early diagnosis. Compared with the traditional methods, nucleic acid amplification technologies are normally free of the pathogen culture process and able to detect pathogens during the incubation period, exhibiting excellent specificity and sensitivity [16,17]. Thus, these technologies are more desirable for early diagnosis, among which real-time polymerase chain reaction (real-time PCR) is the most widely used in clinical diagnosis [18,19].…”

Heat and pressure-resistant room temperature irreversible sealing of hybrid PDMS–thermoplastic microfluidic devices via carbon–nitrogen covalent bonding and its application in a continuous-flow polymerase chain reaction

“…The quantity of DNA can be multiplied in each temperature cycle, which subsequently generates millions of DNA copies between 20 and 35 cycles. Nowadays, the development of miniaturized PCR devices has gained more attention; [19][20][21] in the design of miniaturized PCR devices, continuous-ow-high-throughput PCR (CF-PCR) has many advantages. For instance, the temperature controlling system is much easier than chamber-based PCR, and the PCR mixture is pumped into a straight, 22 serpentine, 23,24 or circular 25,26 microuidic channel through the continuous ow with xed temperature zones.…”