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.

• Keywords
• Electrical and electronic engineering, Microfluidics,
• Publication type
• Journal Article MeSH

• Keywords
• COVID-19, RT-PCR, point of care, point of need, portable systems,
• MeSH
• COVID-19 diagnosis   virology   MeSH
• Equipment Design MeSH
• Humans MeSH
• Point-of-Care Testing MeSH
• RNA, Viral analysis   genetics   MeSH
• SARS-CoV-2 genetics   isolation & purification   MeSH
• Nucleic Acid Amplification Techniques instrumentation   MeSH
• COVID-19 Nucleic Acid Testing instrumentation   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Editorial MeSH
• Names of Substances
• RNA, Viral MeSH

Since its invention in 1986, the polymerase chain reaction (PCR), has become a well-established method for the detection and amplification of deoxyribonucleic acid (DNA) with a specific sequence. Incorporating fluorescent probes, known as TaqMan probes, or DNA intercalating dyes, such as SYBR Green, into the PCR mixture allows real-time monitoring of the reaction progress and extraction of quantitative information. Previously reported real-time PCR product detection using intercalating dyes required melting curve analysis (MCA) to be performed following thermal cycling. Here, we propose a technique to perform dynamic MCA during each thermal cycle, based on a continuous fluorescence monitoring method, providing qualitative and quantitative sample information. We applied the proposed method in multiplexing detection of hepatitis B virus DNA and complementary DNA of human immunodeficiency virus as well as glyceraldehyde 3-phosphate dehydrogenase in different concentration ratios. We extracted the DNA melting curve and its derivative from each PCR cycle during the transition from the elongation to the denaturation temperature with a set heating rate of 0.8 K·s-1and then used the data to construct individual PCR amplification curves for each gene to determine the initial concentration of DNA in the sample. Our proposed method allows researchers to look inside the PCR in each thermal cycle, determining the PCR product specificity in real time instead of waiting until the end of the PCR. Additionally, the slow transition rate from elongation to denaturation provides a dynamic multiplexing assay, allowing the detection of at least three genes in real time.

• Publication type
• Journal Article MeSH

PCR has become one of the most valuable techniques currently used in bioscience, diagnostics and forensic science. Here we review the history of PCR development and the technologies that have evolved from the original PCR method. Currently, there are two main areas of PCR utilization in bioscience: high-throughput PCR systems and microfluidics-based PCR devices for point-of-care (POC) applications. We also discuss the commercialization of these techniques and conclude with a look into their modifications and use in innovative areas of biomedicine. For example, real-time reverse transcription PCR is the gold standard for SARS-CoV-2 diagnoses. It could also be used for POC applications, being a key component of the sample-to-answer system.

• Keywords
• COVID-19, PCR, RNA virus diagnoses, digital PCR, microfluidics, point-of-care diagnostics, portable systems, reverse transcription PCR,
• MeSH
• Betacoronavirus genetics   isolation & purification   MeSH
• COVID-19 MeSH
• Equipment Design MeSH
• Clinical Laboratory Techniques instrumentation   methods   MeSH
• Coronavirus Infections diagnosis   virology   MeSH
• Humans MeSH
• Microfluidic Analytical Techniques instrumentation   methods   MeSH
• Pandemics MeSH
• Polymerase Chain Reaction instrumentation   methods   MeSH
• SARS-CoV-2 MeSH
• COVID-19 Testing MeSH
• Pneumonia, Viral diagnosis   virology   MeSH
• Point-of-Care Systems MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Infectious diseases, such as the most recent case of coronavirus disease 2019, have brought the prospect of point-of-care (POC) diagnostic tests into the spotlight. A rapid, accurate, low-cost, and easy-to-use test in the field could stop epidemics before they develop into full-blown pandemics. Unfortunately, despite all the advances, it still does not exist. Here, we critically review the limited number of prototypes demonstrated to date that is based on a polymerase chain reaction (PCR) and has come close to fulfill this vision. We summarize the requirements for the POC-PCR tests and then go on to discuss the PCR product-detection methods, the integration of their functional components, the potential applications, and other practical issues related to the implementation of lab-on-a-chip technologies. We conclude our review with a discussion of the latest findings on nucleic acid-based diagnosis.

• Keywords
• COVID-19 diagnoses, Future of PCR, Microfluidics, Miniaturization, Point of care, Polymerase chain reaction,
• Publication type
• Journal Article MeSH
• Review MeSH

The global risk of viral disease outbreaks emphasizes the need for rapid, accurate, and sensitive detection techniques to speed up diagnostics allowing early intervention. An emerging field of microfluidics also known as the lab-on-a-chip (LOC) or micro total analysis system includes a wide range of diagnostic devices. This review briefly covers both conventional and microfluidics-based techniques for rapid viral detection. We first describe conventional detection methods such as cell culturing, immunofluorescence or enzyme-linked immunosorbent assay (ELISA), or reverse transcription polymerase chain reaction (RT-PCR). These methods often have limited speed, sensitivity, or specificity and are performed with typically bulky equipment. Here, we discuss some of the LOC technologies that can overcome these demerits, highlighting the latest advances in LOC devices for viral disease diagnosis. We also discuss the fabrication of LOC systems to produce devices for performing either individual steps or virus detection in samples with the sample to answer method. The complete system consists of sample preparation, and ELISA and RT-PCR for viral-antibody and nucleic acid detection, respectively. Finally, we formulate our opinions on these areas for the future development of LOC systems for viral diagnostics.

• Keywords
• Commercialization, Immunoassays, LOC, Microfluidic, Nucleic acid amplification, Viral detection,
• MeSH
• Biosensing Techniques MeSH
• Equipment Design MeSH
• DNA, Viral analysis   MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Lab-On-A-Chip Devices * MeSH
• Humans MeSH
• Microfluidic Analytical Techniques instrumentation   MeSH
• Nucleic Acids analysis   MeSH
• Virus Diseases diagnosis   MeSH
• Point-of-Care Systems MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• DNA, Viral MeSH
• Nucleic Acids MeSH