BACKGROUND: This study introduces an advanced 8-well loop-mediated isothermal amplification (LAMP) system specifically designed for the automated colorimetric detection of SARS-CoV-2. Incorporating two distinct configurations having either three light-emitting diodes (LEDs) with varying emission wavelengths per well, paired with a photodiode detector, or utilizing white LED illumination with a red, green, and blue (RGB) sensor. The colorimetric LAMP aims to provide a more accessible and rapid diagnostic tool than traditional fluorescence methods due to the system's simplicity. RESULTS: We designed, assembled, and compared two colorimetric home-assembled LAMP systems, the first one based on three LEDs, each with a different color with a photodiode, and the second one having RGB and a white LED, with traditional fluorescence-based LAMP method performed on a commercial qPCR instrument. Results demonstrated that the colorimetric RT-LAMP assays achieved critical threshold time (CT), closely matching the CT value of fluorescence-based detection accomplished by the qPCR instrument. We performed the fundamental experiment employing an identical RNA copy number of 1,570copies·μL-1, getting the CT value of (16.70 ± 0.43) min (mean ± standard deviation from 23 measurements). Then, we also performed different RNA numbers of copies between the highest and lowest RNA contents of ≈ 157,000 copies·μL-1 and ≈ 1570 copies·μL-1, respectively, getting CT values from (13.30 ± 0.04) min to (13.75 ± 0.30) min and (17.04 ± 0.02) min to (17.26 ± 0.02) min, all (mean ± standard deviation from three measurements). The colorimetric systems demonstrated rapid response and precision across varied viral loads while keeping the system simple due to the colorimetric detection method. SIGNIFICANCE AND NOVELTY: The LAMP system's rapid and precise detection capabilities underscore its potential as an effective tool for point-of-need diagnostics. It is crucial for timely responses in ongoing and future pandemic scenarios. This system enhances testing accessibility and provides a robust platform for potential adaptation to other pathogenic threats, making it a valuable asset in global health diagnostics.
- Klíčová slova
- Biosensors and actuators, Colorimetric detection, Loop-mediated isothermal amplification (LAMP), Point-of-care diagnostics, Rapid testing technologies, SARS-CoV-2 diagnostics,
- MeSH
- COVID-19 * diagnóza virologie MeSH
- diagnostické techniky molekulární metody MeSH
- kolorimetrie * metody MeSH
- lidé MeSH
- RNA virová analýza genetika MeSH
- SARS-CoV-2 * genetika izolace a purifikace MeSH
- techniky amplifikace nukleových kyselin * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- RNA virová MeSH
This study presents a rapid and versatile low-cost sample-to-answer system for SARS-CoV-2 diagnostics. The system integrates the extraction and purification of nucleic acids, followed by amplification via either reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or reverse transcription loop-mediated isothermal amplification (RT-LAMP). By meeting diverse diagnostic and reagent needs, the platform yields testing results that closely align with those of commercial RT-LAMP and RT‒qPCR systems. Notable advantages of our system include its speed and cost-effectiveness. The assay is completed within 28 min, including sample loading (5 min), ribonucleic acid (RNA) extraction (3 min), and RT-LAMP (20 min). The cost of each assay is ≈ $9.5, and this pricing is competitive against that of Food and Drug Administration (FDA)-approved commercial alternatives. Although some RNA loss during on-chip extraction is observed, the platform maintains a potential limit of detection lower than 297 copies. Portability makes the system particularly useful in environments where centralized laboratories are either unavailable or inconveniently located. Another key feature is the platform's versatility, allowing users to choose between RT‒qPCR or RT‒LAMP tests based on specific requirements.
- Klíčová slova
- Chemistry, Electrical and electronic engineering, Microfluidics,
- Publikační typ
- časopisecké články MeSH
This study elaborates on the design, fabrication, and data analysis details of SPEED, a recently proposed smartphone-based digital polymerase chain reaction (dPCR) device. The dPCR chips incorporate partition diameters ranging from 50 μm to 5 μm, and these partitions are organized into six distinct blocks to facilitate image processing. Due to the superior thermal conductivity of Si and its potential for mass production, the dPCR chips were fabricated on a Si substrate. A temperature control system based on a high-power density Peltier element and a preheating/cooling PCR protocol user interface shortening the thermal cycle time. The optical design employs four 470 nm light-emitting diodes as light sources, with filters and mirrors effectively managing the light emitted during PCR. An algorithm is utilized for image processing and illumination nonuniformity correction including conversion to a monochromatic format, partition identification, skew correction, and the generation of an image correction mask. We validated the device using a range of deoxyribonucleic acid targets, demonstrating its potential applicability across multiple fields. Therefore, we provide guidance and verification of the design and testing of the recently proposed SPEED device.
- Klíčová slova
- Electrical and electronic engineering, Microfluidics,
- Publikační typ
- časopisecké články MeSH
We demonstrate a smartphone integrated handheld (SPEED) digital polymerase chain reaction (dPCR) device for point-of-care application. The device has dimensions of ≈100 × 200 × 35 mm3 and a weight of ≈400 g. It can perform 45 PCR cycles in ≈49 min. The device also features integrated, miniaturized modules for thermal cycling, image taking, and wireless data communication. These functions are controlled by self-developed Android-based applications. The only consumable is the developed silicon-based dPCR chip, which has the potential to be recycled. The device's precision and accuracy are comparable with commercial dPCR machines. We have verified the SPEED dPCR prototype's utility in the testing of severe acute respiratory syndrome coronavirus 2, the detection of cancer-associated gene sequences, and the confirmations of Down syndrome diagnoses. Due to its low upfront capital investment, as well as its nominal running cost, we envision that the SPEED dPCR device will help to perform cancer screenings and non-invasive prenatal tests for the general population. It will also aid in the timely identification and monitoring of infectious disease testing, thereby expediting alerts with respect to potential emerging pandemics.
- MeSH
- biosenzitivní techniky * MeSH
- chytrý telefon MeSH
- COVID-19 * diagnóza MeSH
- lidé MeSH
- nádory * MeSH
- polymerázová řetězová reakce MeSH
- testování na COVID-19 MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
