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Droplet microfluidic systems have emerged as indispensable and advanced tools in contemporary biological science. A prominent example is the droplet digital polymerase chain reaction (ddPCR), which plays a pivotal role in next-generation sequencing and the detection of rare nucleic acids or mutations. However, existing optical detection configurations are bulky, intricate, and costly, and require meticulous optical alignment to optimize fluorescence sensing. Herein, we propose a lab-in-fiber optofluidic system (LiFO), which provides a stable and compact footprint, self-alignment, and enhanced optical coupling for high-accuracy ddPCR. Moreover, LiFO could expand its capabilities for multiangle-scattering light collection in which we collect focused forward-scattering light (fFSL) to enable real-time droplet counting and size monitoring. To accomplish these attributes, LiFO incorporates optical fibers, along with fabricated PDMS grooves, for a self-aligned optical setup to implement simultaneous fluorescence and scattering detection. Furthermore, LiFO harnesses the concept of flowing droplets functioning as microlenses, which allows us to collect and translate fFSL signals into droplet size information. We have demonstrated the effectiveness of LiFO in ddPCR applications, illustrating its capacity to enhance the accuracy and precision of DNA quantification. Notably, LiFO exhibits improved linearity in the measurement of serial DNA dilutions, reflected by an increase in R 2 from 0.956 to 0.997. These results demonstrate the potential of LiFO to serve as a valuable tool across a wide spectrum of droplet microfluidic platforms, offering opportunities for advancement in practical applications.

Droplet digital PCR (ddPCR) is recognized as a high-precision method for nucleic acid quantification, extensively utilized in biomedical research and clinical diagnostics. This technique employs microfluidic technology to partition the nucleic acid-containing reaction mixture into discrete droplets for amplification, achieving absolute quantification by identifying and enumerating the number of fluorescent droplets. The accuracy of droplet quantification is pivotal to the success of the assay. However, current image-processing tools are operationally complex, and commercial instruments are costly. Moreover, the designed algorithms exhibit a need for enhanced accuracy and are often restricted to use by trained personnel with specific microscopy equipment. In response to these challenges, we introduce an automated device (A-MMD), capable of detecting fluorescent droplets in ddPCR images captured by multiple microscopes. The device integrates three distinct algorithms tailored for the image processing of Laser Scanning Confocal Microscopy (LSCM), inverted microscopy, and self-assembled microscopy. Experimental validation using λ DNA demonstrated a 100.00% identification rate for positive droplets across all three image types, and the average identification rates for total droplets being 99.27% for LSCM, 98.96% for inverted microscopy, and 99.08% for self-assembled microscopy. Furthermore, the A-MMD is equipped with a user-friendly interface (UI) that streamlines the operational process, enabling non-specialists to efficiently perform droplet detection tasks. Our device not only has good environmental adaptability and identification accuracy, but also significantly reduces costs and operational complexity. It offers an economical, efficient, and user-friendly solution for ddPCR image analysis, thereby further propelling the advancement and application of nucleic acid detection technology.

Droplet digital polymerase chain reaction (ddPCR) stands out as a highly sensitive diagnostic technique that is gaining traction in infectious disease diagnostics due to its ability to quantitate very low numbers of viral gene copies. By partitioning the sample into thousands of droplets, ddPCR enables precise and absolute quantification without relying on a standard curve. However, current ddPCR systems often exhibit relatively low levels of integration, and the analytical process remains dependent on elaborate workflows for up-front sample preparation. Here, we introduce a fully-integrated system seamlessly combining viral lysis, RNA extraction, emulsification, reverse transcription (RT) ddPCR, and fluorescence readout in a sample-to-answer format. The system comprises a disposable microfluidic cartridge housing buffers and reagents required for the assay, and a centrifugal platform that allows for pneumatic actuation of liquids during rotation, enabling automation of the workflow. Highly monodisperse droplets (∼50 μm in diameter) are produced using centrifugal step emulsification and automatically transferred to an integrated heating module for target amplification. The platform is equipped with a miniature fluorescence imaging system enabling on-chip read-out of droplets after RT-ddPCR. We demonstrate sample-to-answer detection of SARS-CoV-2 N and E genes, along with RNase P endogenous reference, using hydrolysis probes and multiplexed amplification within single droplets for concentrations as low as 0.1 copy per μL. We also tested 14 nasopharyngeal swab specimens from patients and were able to distinguish positive and negative SARS-CoV-2 samples with 100% accuracy, surpassing results obtained by conventional real-time amplification.

Polymerase chain reaction (PCR) is the gold standard for nucleic acid amplification and quantification in diverse fields such as life sciences, global health, medicine, agricultural science, forensic science, and environmental science for global sustainability. However, implementing a cost-effective PCR remains challenging for rapid preventive medical action to the widespread pandemic diseases due to the absence of highly efficient and low-cost PCR chip-based POC molecular diagnostics. Here, this work reports an ultrafast metaphotonic PCR chip as a solution of a cost-effective and low-power-consumption POC device for the emerging global challenge of sustainable healthcare. This work designs a near-perfect photonic meta-absorber using ring-shaped titanium nitride to maximize the photothermal effect and realize rapid heating and cooling cycles during the PCR process. This work fabricates a large-area photonic meta-absorber on a 6-inch wafer cost-effectively using simple colloidal lithography. In addition, this work demonstrates 30 thermocycles from 65 (annealing temperature) to 95 °C (denaturation temperature) within 3 min 15 s, achieving an average 16.66 °C s -1 heating rate and 7.77 °C s -1 cooling rate during thermocycling, succeeding rapid metaphotonic PCR. This work believes a metaphotonic PCR chip can be used to create a low-cost, ultrafast molecular diagnostic chip with a meta-absorber., (© 2024 The Author(s). Advanced Materials published by Wiley‐VCH GmbH.)

Viral RNA detection is crucial in preventing and treating early infectious diseases. Traditional methods of RNA detection require a large amount of equipment and technical personnel. In this study, proposed a programmable controlled surface acoustic wave (SAW)-based RNA detecting device has been proposed. The proposed device can perform the entire viral RNA detection process, including cell lysis by cell-microparticle collision through SAW-induced liquid whirling, RNA capture by SAW-suspended magnetic beads, RNA elution through SAW-induced high streaming force, and PCR thermal cycling through SAW-generated heat. The device has completed all RNA detection steps in one mini chamber, requiring only 489 µl reagents for RNA extraction, much smaller than the amount used in manual RNA extraction (2065 µl). The experimental results have shown that PCR results from the device are comparable to those achieved via commercial qPCR instrumental detection. This work has demonstrated the potential of SAW-based lab-on-a-chip devices for point-of-care testing and provided a novel approach for rapidly detecting infectious diseases., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

High-cost DNA extraction procedures pose significant challenges for budget-constrained laboratories. To address this, we introduce OpenCell, an economical, open-source, 3-in-1 laboratory device that combines the functionalities of a bead homogenizer, a microcentrifuge, and a vortex mixer. OpenCell utilizes modular attachments that magnetically connect to a central rotating brushless motor. This motor couples to an epicyclic gearing mechanism, enabling efficient bead homogenization, vortex mixing, and centrifugation within one compact unit. OpenCell's design incorporates multiple redundant safety features, ensuring both the device's and operator's safety. Additional features such as RPM measurement, programmable timers, battery operation, and optional speed control make OpenCell a reliable and reproducible laboratory instrument. In our study, OpenCell successfully isolated DNA from Spinacia oleracea (spinach), with an average yield of 2.3 μg and an A260/A280 ratio of 1.77, demonstrating its effectiveness for downstream applications such as Polymerase Chain Reaction (PCR) amplification. With its compact size (20 cm x 28 cm x 6.7 cm) and lightweight design (0.8 kg), comparable to the size and weight of a laptop, OpenCell is portable, making it an attractive component of a 'lab-in-a-backpack' for resource-constrained environments in low-and-middle-income countries and synthetic biology in remote field stations. Leveraging the accessibility of 3D printing and off-the-shelf components, OpenCell can be manufactured and assembled at a low unit cost of less than $50, providing an affordable alternative to expensive laboratory equipment costing over $4000. OpenCell aims to overcome the barriers to entry in synthetic biology research and contribute to the growing collection of frugal and open hardware., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gupta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases. Analysis of DNA methylation patterns has until now been dependent on either a chemical or an enzymatic pre-treatment, which are both time consuming procedures and potentially biased due to incomplete treatment. We present a qPCR technology, EpiDirect®, that allows for direct PCR quantification of DNA methylations using untreated DNA. EpiDirect® is based on the ability of Intercalating Nucleic Acids (INA®) to differentiate between methylated and unmethylated cytosines in a special primer design. With this technology, we develop an assay to analyze the methylation status of a region of the MGMT promoter used in treatment selection and prognosis of glioblastoma patients. We compare the assay to two bisulfite-relying, methyl-specific PCR assays in a study involving 42 brain tumor FFPE samples, revealing high sensitivity, specificity, and the clinical utility of the method., (© 2023. Springer Nature Limited.)

Droplet digital polymerase chain reaction (ddPCR) is a new quantitative PCR method based on water-oil emulsion droplet technology. ddPCR enables highly sensitive and accurate quantification of nucleic acid molecules, especially when their copy numbers are low. In ddPCR, a sample is fractionated into ~20,000 droplets, and every nanoliter-sized droplet undergoes PCR amplification of the target molecule. The fluorescence signals of droplets are then recorded by an automated droplet reader. Circular RNAs (circRNAs) are single-stranded, covalently closed RNA molecules that are ubiquitously expressed in animals and plants. CircRNAs are promising as biomarkers for cancer diagnosis and prognosis and as therapeutic targets or agents to inhibit oncogenic microRNAs or proteins (Kristensen LS, Jakobsen T, Hager H, Kjems J, Nat Rev Clin Oncol 19:188-206, 2022). In this chapter, the procedures for the quantitation of a circRNA in single pancreatic cancer cells using ddPCR are described., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Molecular biology is a widely used and widespread technique in research and as a laboratory diagnostic tool, aiming to investigate targets of interest from the obtainment, identification, and analysis of genetic material. In this context, methods, such as Polymerase Chain Reaction (PCR), Reverse Transcription Polymerase Chain Reaction (RT-PCR), real-time PCR, loopmediated isothermal amplification (LAMP), and loop-mediated isothermal amplification with reverse transcription (RT-LAMP), can be cited. Such methods use enzymes, buffers, and thermosensitive reagents, which require specific storage conditions. In an attempt to solve this problem, the lyophilization procedure (dehydration process by sublimation) can be applied, aiming to preserve and prolong the useful life of the reaction components in cases of temperature variation. In this review, we present a synthesis of the lyophilization process, describing the events of each step of the procedure and providing general information about the technique. Moreover, we selected lyophilization protocols found in the literature, paying attention to the conditions chosen by the authors for each step of the procedure, and structured the main data in tables, facilitating access to information for researchers who need material to produce new functional protocols., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The outbreak of COVID-19 pandemic and its consequences have inflicted a substantial damage on the world. In this study, it was attempted to review the recent coronaviruses appeared among the human being and their epidemic/pandemic spread throughout the world. Currently, there is an inevitable need for the establishment of a quick and easily available biosensor for tracing COVID-19 in all countries. It has been known that the incubation time of COVID-19 lasts about 14 days and 25% of the infected individuals are asymptomatic. To improve the ability to determine SARS-CoV-2 precisely and reduce the risk of eliciting false-negative results produced by mutating nature of coronaviruses, many researchers have established a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using mismatch-tolerant molecular beacons as multiplex real-time RT-PCR to distinguish between pathogenic and non-pathogenic strains of coronaviruses. The possible mechanisms and pathways for the detection of coronaviruses by biosensors have been reviewed in this study., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Polymerase chain reaction (PCR) is the most widely used method for nucleic acids amplification. To date, a huge number of versatile PCR techniques have been developed. One of the relevant goals is to shorten PCR duration, which can be achieved in several ways. Here, we report on the results regarding nucleic acids amplification by convective PCR (cPCR) in standard 0.2 ml polypropylene microtubes. The following conditions were found to be optimal for such amplification: 1) 70 μl reaction volume, 2) the supply of external temperature 145°С for the denaturation zone and 0°С for the annealing zone, 3) ∼30° inclination of the microtube main axis, 4) the use of nearby primers, and 5) duration of the reaction 15-20 min. At these conditions, the amplification products are accumulated in an amount sufficient to be registered by gel electrophoresis, and high sensitivity of the reaction comparable to that of conventional PCR is achieved. cPCR provided the reliable detection of SARS-CoV-2 coronavirus RNA isolated from nasopharyngeal swabs of COVID-19 patients., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Reverse transcription PCR (RT-PCR) is a popular method for detecting RNA viruses in plants. RT-PCR is usually performed in a classical two-step procedure: in the first step, cDNA is synthesized by reverse transcriptase (RT), followed by PCR amplification by a thermostable polymerase in a separate tube in the second step. However, one-step kits containing multiple enzymes optimized for RT and PCR amplification in a single tube can also be used. Here, we describe an RT-PCR single-enzyme assay based on an RTX DNA polymerase that has both RT and polymerase activities. The expression plasmid pET_RTX_(exo-) was transferred to various E. coli genotypes that either compensated for codon bias (Rosetta-gami 2) or contained additional chaperones to promote solubility (BL21 (DE3) with plasmids pKJE8 or pTf2). The RTX enzyme was then purified and used for the RT-PCR assay. Several purified plant viruses (TMV, PVX, and PVY) were used to determine the efficiency of the assay compared to a commercial one-step RT-PCR kit. The RT-PCR assay with the RTX enzyme was validated for the detection of viruses from different genera using both total RNA and crude sap from infected plants. The detection endpoint of RTX-PCR for purified TMV was estimated to be approximately 0.01 pg of the whole virus per 25 µL reaction, corresponding to 6 virus particles/µL. Interestingly, the endpoint for detection of TMV from crude sap was also 0.01 pg per reaction in simulated crude plant extracts. The longest RNA fragment that could be amplified in a one-tube arrangement was 2379 bp long. The longest DNA fragment that could be amplified during a 10s extension was 6899 bp long. In total, we were able to detect 13 viruses from 11 genera using RTX-PCR. For each virus, two to three specific fragments were amplified. The RT-PCR assay using the RTX enzyme described here is a very robust, inexpensive, rapid, easy to perform, and sensitive single-enzyme assay for the detection of plant viruses.

The real-time reverse transcription-polymerase chain reaction (real-time RT-qPCR) has become a leading technique for the detection and quantification of arboviruses, including Chikungunya, Dengue, and Zika viruses. In this study, an updated real-time RT-qPCR assay was designed and evaluated together with a synthetic positive-control chimeric RNA for the simultaneous detection and quantification of Chikungunya, Dengue, and Zika viruses. Amplification assays were performed to verify the construct integrity and optimal reaction/thermal cycling conditions. The analytical sensitivity of the assay was determined for each virus in single and multiplex reactions, as well as the performance in the detection and viral load quantification of experimental samples. The real-time RT-qPCR assay presented here allowed for the simultaneous detection and quantification of Chikungunya, Dengue, and Zika viruses and could be applied in several studies where the accurate quantification of viral genomes is required., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Assays for the molecular detection of nucleic acids are typically constrained by the level of multiplexing (this is the case for the quantitative polymerase chain reaction (qPCR) and for isothermal amplification), turnaround times (as with microarrays and next-generation sequencing), quantification accuracy (isothermal amplification, microarrays and nanopore sequencing) or specificity for single-nucleotide differences (microarrays and nanopore sequencing). Here we show that a portable and battery-powered PCR assay performed in a toroidal convection chamber housing a microarray of fluorescently quenched oligonucleotide probes allows for the rapid and sensitive quantification of multiple DNA targets with single-nucleotide discrimination. The assay offers a limit of detection of 10 DNA copies within 30 min of turnaround time and a dynamic range spanning 4 orders of magnitude of DNA concentration, and we show its performance by detecting 20 genomic loci and 30 single-nucleotide polymorphisms in human genomic DNA samples, and 15 bacterial species in clinical isolates. Portable devices for the fast and highly multiplexed detection of nucleic acids may offer advantages in point-of-care diagnostics., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Molecular diagnostics has been the front runner in the world's response to the COVID-19 pandemic. Particularly, reverse transcriptase-polymerase chain reaction (RT-PCR) and the quantitative variant (qRT-PCR) have been the gold standard for COVID-19 diagnosis. However, faster antigen tests and other point-of-care (POC) devices have also played a significant role in containing the spread of SARS-CoV-2 by facilitating mass screening and delivering results in less time. Thus, despite the higher sensitivity and specificity of the RT-PCR assays, the impact of POC tests cannot be ignored. As a consequence, there has been an increased interest in the development of miniaturized, high-throughput, and automated PCR systems, many of which can be used at point-of-care. This review summarizes the recent advances in the development of miniaturized PCR systems with an emphasis on COVID-19 detection. The distinct features of digital PCR and electrochemical PCR are detailed along with the challenges. The potential of CRISPR/Cas technology for POC diagnostics is also highlighted. Commercial RT-PCR POC systems approved by various agencies for COVID-19 detection are discussed.

Background: Copy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum plasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with anti-malarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to anti-malarial drugs., Methods: A multiplex ddPCR assay was developed to detect the CNVs in the pfmdr1 and pfplasmepsin2 genes, while a duplex ddPCR assay was developed to detect CNV in the pfgch1 gene. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In order to reduce the cost of testing, a multiplex ddPCR assay of two target genes, pfmdr1 and pfplasmepsin2, was validated. In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay., Results: There were no significant differences between the GCN results obtained from uniplex and multiplex ddPCR assays for detection of CNVs in the pfmdr1 and pfplasmepsin2 genes (p = 0.363 and 0.330, respectively). Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. There was no significant difference in gene copy numbers assessed by uniplex or duplex ddPCR assays regarding CNV in the pfgch1 gene (p = 0.276). The accuracy and %RSD value of the duplex ddPCR assay were 95% and 4%, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). The results suggested that multiplex ddPCR assay is the optional assay for the accurate detection of gene copy number without requiring calibration standards, while the cost and required time are reduced. Based on the results of this study, criteria for GCN detection by ddPCR analysis were generated., Conclusions: The developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes of P. falciparum. The ddPCR assay is a useful additional tool for the surveillance of anti-malarial drug resistance.

Nucleic acid amplification tests for diagnosing Clostridioides difficile infections (CDI) are improving to become faster and more accurate. This study aimed to evaluate the accuracy of rapid detection of toxigenic C. difficile using the novel high-speed polymerase chain reaction (PCR) device, PathOC RightGene. These results were compared and evaluated with real-time PCR (qPCR) and enzyme immunoassays (EIA) kit. For this study, 102 C. difficile and 3 Clostridium species isolated from CDI patients were used. These C. difficile isolates were 85 toxigenic and 17 non-toxigenic strains. The results of qPCR served as a standard, and sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the PathOC Right Gene were 99.2%, 99.4%, 100%, 98.8%, and 99.3%, respectively. Turnaround time of qPCR and EIA was 85 and 30 minutes, whereas PathOC RightGene was only 25 minutes including DNA extraction. This novel high-speed PCR device detected toxigenic C. difficile rapidly and accurately., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Molecular diagnostics has become essential in the identification of many infectious and neglected diseases, and the detection of nucleic acids often serves as the gold standard technique for most infectious agents. However, established techniques like polymerase chain reaction (PCR) are time-consuming laboratory-bound techniques while rapid tests such as Lateral Flow Immunochromatographic tests often lack the required sensitivity and/or specificity., Methods/principle Findings: Here we present an affordable, highly mobile alternative method for the rapid identification of infectious agents using pulse-controlled amplification (PCA). PCA is a next generation nucleic acid amplification technology that uses rapid energy pulses to heat microcyclers (micro-scale metal heating elements embedded directly in the amplification reaction) for a few microseconds, thus only heating a small fraction of the reaction volume. The heated microcyclers cool off nearly instantaneously, resulting in ultra-fast heating and cooling cycles during which classic amplification of a target sequence takes place. This reduces the overall amplification time by a factor of up to 10, enabling a sample-to-result workflow in just 15 minutes, while running on a small and portable prototype device. In this proof of principle study, we designed a PCA-assay for the detection of Yersinia pestis to demonstrate the efficacy of this technology. The observed detection limits were 434 copies per reaction (purified DNA) and 35 cells per reaction (crude sample) respectively of Yersinia pestis., Conclusions/significance: PCA offers fast and decentralized molecular diagnostics and is applicable whenever rapid, on-site detection of infectious agents is needed, even under resource limited conditions. It combines the sensitivity and specificity of PCR with the rapidness and simplicity of hitherto existing rapid tests., Competing Interests: I have read the journal's policy and the authors SD, LU, JS and FB of this manuscript have the following competing interests: SD is working for GNA biosolutions. LU, JS and FB are the founders and CEOs of GNA biosolution.

In New Zealand, a new diagnostic approach for the control of paratuberculosis in mixed aged milking cows has been developed using a combination of ELISA and quantitative fecal PCR (f-qPCR). Our analysis was designed to evaluate performance of these individual tests in infected or infectious mixed aged cows across the prevalence of infection typically encountered on NZ dairy farms and calculate test accuracy when used as a screening test of serological ELISAs for four separate antigens read in parallel followed by a confirmatory quantitative f-qPCR test. Data from a cross-sectional study of 20 moderate prevalence herds was combined with existing data from 2 low and 20 high prevalence herds forming a dataset of 3845 paired serum and fecal samples. Incidence of clinical Johne's disease (JD) was used to classify herds into three prevalence categories. High (≥ 3% annual clinical JD for the last three years), moderate (<3 - 1%) and low (<1% incidence for at least the last five years). Positive tests were declared if> 50 ELISA units and f-qPCR at two cut-points (≥1 × 10 4 genomes/mL or >1 × 10 3 genomes/mL). Fixed Bayesian latent class models at both f-qPCR cut-points, accounted for conditional independence and paired conditional dependence. Mixed models at both f-qPCR cut-points, using a different mechanism to account for conditional dependencies between tests were also implemented. Models (24 in number) were constructed using OpenBUGS. The aim was to identify Mycobacterium avium subsp. paratuberculosis (MAP) infected cows that met at least one of two criteria: shedding sufficient MAP in feces to be detected by f-qPCR or mounting a detectable MAP antibody response. The best fit to the data was obtained by modelling pairwise dependencies between tests in a fixed model or by accounting for dependencies in a mixed model at a fecal cut-off of ≥1 × 10 4 genomes/mL. Test performance differed with prevalence, but models were robust to prior assumptions. For the fixed model, at a prevalence of 0.29 (95 % probability interval (PI) = 0.25-0.33), as a screening plus confirmatory f-qPCR, post-test probability for disease in a positive animal was 0.84 (95 %PI = 0.80-0.88) and 0.16 (95 %PI = 0.15-0.18) for disease in a test negative animal. In low prevalence herds (0.01(95 %PI = 0.00-0.04)) the equivalent figures were 0.84 (95 %PI = 0.08-0.92) and 0.00 (95 %PI = 0.00-0.02). These results suggest this is a useful tool to control JD on dairy farms, particularly in herds with higher levels of infection, where the sampling and testing cost per animal is defrayed across more detected animals., (Copyright © 2020 Elsevier B.V. All rights reserved.)

There is a need for novel analytical techniques to study the composition of single extracellular vesicles (EV). Such techniques are required to improve the understanding of heterogeneous EV populations, to allow identification of unique subpopulations, and to enable earlier and more sensitive disease detection. Because of the small size of EV and their low protein content, ultrahigh sensitivity technologies are required. Here, an immuno-droplet digital polymerase chain reaction (iddPCR) amplification method is described that allows multiplexed single EV protein profiling. Antibody-DNA conjugates are used to label EV, followed by stochastic microfluidic incorporation of single EV into droplets. In situ PCR with fluorescent reporter probes converts and amplifies the barcode signal for subsequent read-out by droplet imaging. In these proof-of-principle studies, it is shown that multiplex protein analysis is possible in single EV, opening the door for future analyses., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

With the rapid development of digital precision medicine, the digital polymerase chain reaction (dPCR) deoxyribonucleic acid (DNA) gene chip integrates more channels with smaller size and larger area, which leads to a higher technical requirement for commercial optical fluorescence microscopy. The multitime image splicing method is widely used for DNA detection. However, it consumes time and has visible seamless image results. This work has demonstrated the design and fabrication of a three channel reversed and reduced fluorescence microscopic imaging system with high-resolution and large field of view for one-time imaging. We introduced the super ultra-thin dichroic mirror into the space between the objective lens and the gene chip to achieve a uniform illumination and a strong signal for the large area gene chip. The fabricated new fluorescence microscopy can take a one-time imaging for the 28×18 m m dPCR gene chip with more than 20,000 multi micro-droplets within FAM, HEX, and ROX fluorescence channels. The optical system was designed with a numerical aperture (NA) of 0.106. Modulation transfer function (MTF) is higher than 0.675 at 70 lp/mm, and the function resolution capability is 10 µm with the whole magnification of -0.65 t i m e s . The fly's eye lens-based illumination system was tested with a uniform output of over 90% in the whole ϕ 34.7 m m chip area. The design was tested, and the experimental results showed that this new system provides a fast, efficient, and professional optical imaging method for detection of the new emerged digital PCR gene chip, which has larger area and more channels.

This paper presents a digital PCR system based on a novel thermal cycled chip, which wraps microchannels on a trapezoidal structure made of polydimethylsiloxane (PDMS) in a multi-helix manner for the first time. It is found that compared to the single helix chip commonly used in previous reports, this kind of novel multi-helix chip can make the surface temperature in the renaturation zone more uniform, and even in the case of rapid fluid flow, it can improve the efficiency of the polymerase chain reaction. What's more, the winding method of multi helix (such as double helix, six helix and eight helix) can obtain better temperature uniformity than the winding of odd helix (such as single helix and three helix). As a proof of concept, the temperature-optimized double-helical chip structure is applied to continuous-flow digital PCR and there is no need to add any surfactant to both the oil phase and reagent. In addition, we successfully analyzed the fluorescence signal of continuous-flow digital PCR by using CMOS camera. Finally, this method is applied for the absolute quantification of the clinical serum sample infected by HBV. The accuracy of the test results has been confirmed by commercial instruments.

A miniaturized polymerase chain reaction (PCR) system is not only important for medical applications in remote areas of developing countries, but also important for testing at ports of entry during global epidemics, such as the current outbreak of the coronavirus. Although there is a large number of PCR sensor systems available for this purpose, there is still a lack of portable digital PCR (dPCR) heating systems. Here, we first demonstrated a portable plasmonic heating-based dPCR system. The device has total dimensions of 9.7 × 5.6 × 4.1 cm and a total power consumption of 4.5 W, allowing for up to 25 dPCR experiments to be conducted on a single charge of a 20 000 mAh external battery. The dPCR system has a maximum heating rate of 10.7 °C s -1 and maximum cooling rate of 8 °C s -1 . Target DNA concentrations in the range from 101 ± 1.4 copies per μL to 260 000 ± 20 000 copies per μL could be detected using a poly(dimethylsiloxane) (PDMS) microwell membrane with 22 080 well arrays (20 μm diameter). Furthermore, the heating system was demonstrated using a mass producible poly(methyl methacrylate) PMMA microwell array with 8100 microwell arrays (80 μm diameter). The PMMA microwell array could detect a concentration from 12 ± 0.7 copies per μL to 25 889 ± 737 copies per μL.

Timely detection and diagnosis are urgently needed to guide epidemiological measures, infection control, antiviral treatment, and vaccine research. In this review, biomarkers/indicators for diagnosis of coronavirus disease 2019 (COVID-19) or detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the environment are summarized and discussed. It is concluded that the detection methods targeting antibodies are not suitable for screening of early and asymptomatic cases since most patients had an antibody response at about 10 days after onset of symptoms. However, antibody detection methods can be combined with quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) to significantly improve the sensitivity and specificity of diagnosis, and boost vaccine research. Fast, sensitive and accurate detection methods targeting antigens need to be developed urgently. Various specimens for diagnosis or detection are compared and analyzed. Among them, deep throat saliva and induced sputum are desired for RT-qPCR test or other early detection technologies. Chest computerized tomography (CT) scan, RT-qPCR, lateral flow immunochromatographic strip (LFICS) for diagnosis of COVID-19 are summarized and compared. Specially, potential electrochemical (EC) biosensor, surface enhanced Raman scattering (SERS)-based biosensor, field-effect transistor (FET)-based biosensor, surface plasmon resonance (SPR)-based biosensor and artificial intelligence (AI) assisted diagnosis of COVID-19 are emphasized. Finally, some commercialized portable detection device, current challenges and future directions are discussed., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The clinical and epidemiologic management of the SARS-CoV-2 pandemic is critically dependent on molecular assays with short turn-around time. We validated the novel Xpert Xpress SARS-CoV-2 assay using a commercial nucleic acid testing (Roche Cobas 6800). We found an excellent concordance over a range of SARS-CoV-2 loads and across established human coronaviruses., (Copyright © 2020. Published by Elsevier B.V.)

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.

While the FilmArray Respiratory Panel EZ has been proven to reduce inappropriate antibiotic use in the outpatient pediatric setting, it is unclear whether its implementation will also reduce downstream health costs such as provider visits and telephone calls. This analysis will help pediatricians make more informed decisions on the implementation and judicious use of the Respiratory Panel EZ in their clinical practice.

Existing digital polymerase chain reaction (PCR) devices consist of multiple independent devices, such as a droplet generator, a PCR thermocycler, and a droplet reader, which have the disadvantages of low integration, complex equipment structure, and high operation difficulty. This paper proposes a fully automatic integrated digital PCR device based on continuous-flow digital PCR theory. By simply adding the sample to the entrance of the integrated instrument, a series of procedures required for digital PCR detection can be fully automated, including sample injection, droplet generation, PCR thermal cycling, fluorescence acquisition, and signal analysis. In contrast to traditional techniques, sample testing requires only one integrated device rather than three separate instruments. For full automation, we design complete control and data processing software, which can complete a test by one-step operation. Therefore, the disadvantages of traditional instruments, such as multi-step operation, and hence, potential environmental pollution, are avoided. Moreover, the system can be powered by solar cells and does not require an external power supply. As a proof of concept, the proposed device is used for absolute quantitative detection of the hepatitis B virus in serum samples. The capacity of the system is validated by absolute quantification of three orders of magnitude from 10 3 to 10 5 IU/mL. The results have a good linear correlation (0.9986) with those of the traditional quantitative (qPCR), thus confirming the reliability of the instrument. In summary, we believe that our work can promote the development of integrated digital PCR systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The coronavirus disease 2019 (COVID-19) pandemic has crudely demonstrated the need for massive and rapid diagnostics. By the first week of July, more than 10,000,000 positive cases of COVID-19 have been reported worldwide, although this number could be greatly underestimated. In the case of an epidemic emergency, the first line of response should be based on commercially available and validated resources. Here, we demonstrate the use of the miniPCR, a commercial compact and portable PCR device recently available on the market, in combination with a commercial well-plate reader as a diagnostic system for detecting genetic material of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19. We used the miniPCR to detect and amplify SARS-CoV-2 DNA sequences using the sets of initiators recommended by the World Health Organization (WHO) for targeting three different regions that encode for the N protein. Prior to amplification, samples were combined with a DNA intercalating reagent (i.e., EvaGreen Dye). Sample fluorescence after amplification was then read using a commercial 96-well plate reader. This straightforward method allows the detection and amplification of SARS-CoV-2 nucleic acids in the range of ~625 to 2×105 DNA copies. The accuracy and simplicity of this diagnostics strategy may provide a cost-efficient and reliable alternative for COVID-19 pandemic testing, particularly in underdeveloped regions where RT-QPCR instrument availability may be limited. The portability, ease of use, and reproducibility of the miniPCR makes it a reliable alternative for deployment in point-of-care SARS-CoV-2 detection efforts during pandemics., Competing Interests: The authors have declared that no competing interests exist.

Monosaccharide composition analysis after acid hydrolysis is the first step towards structural characterization of the polysaccharides. To modernize the hydrolytic procedure, we used a polymerase chain reaction (PCR) instrument to accomplish the task, which allows to generate monosaccharide products from up to 96 samples simultaneously within 30 min. Fucoidan, chitosan and propylene glycol alginate sodium sulfate (PSS) were chosen as representatives of complex, basic and acidic polysaccharides to optimize the hydrolytic conditions, respectively, through the orthogonal L9 (3 4 ) experiments. The hydrolysis loss ratio for monosaccharide standards were also measured. Using this assay, the hydrolysis plus 1-phenyl-3-methyl-5-pyrazolone (PMP) labeling of the monosaccharide products could be accomplished in 90 min with the RSD values less than 5 % based on HPLC analysis. We further confirmed the reliability of the assay by HPLC coupled MS analysis. In conclusion, PCR instrument-based hydrolysis assay is suitable for monosaccharide composition analysis of complex, acidic and basic polysaccharides., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest with content of the manuscript., (Copyright © 2020. Published by Elsevier Ltd.)

Background: Plasmodium knowlesi and Plasmodium vivax are the predominant Plasmodium species that cause malaria in Malaysia and play a role in asymptomatic malaria disease transmission in Malaysia. The diagnostic tools available to diagnose malaria, such as microscopy and rapid diagnostic test (RDT), are less sensitive at detecting lower parasite density. Droplet digital polymerase chain reaction (ddPCR), which has been shown to have higher sensitivity at diagnosing malaria, allows direct quantification without the need for a standard curve. The aim of this study is to develop and use a duplex ddPCR assay for the detection of P. knowlesi and P. vivax, and compare this method to nested PCR and qPCR., Methods: The concordance rate, sensitivity and specificity of the duplex ddPCR assay were determined and compared to nested PCR and duplex qPCR., Results: The duplex ddPCR assay had higher analytical sensitivity (P. vivax = 10 copies/µL and P. knowlesi = 0.01 copies/µL) compared to qPCR (P. vivax = 100 copies/µL and P. knowlesi = 10 copies/µL). Moreover, the ddPCR assay had acceptable clinical sensitivity (P. vivax = 80% and P. knowlesi = 90%) and clinical specificity (P. vivax = 87.84% and P. knowlesi = 81.08%) when compared to nested PCR. Both ddPCR and qPCR detected more double infections in the samples., Conclusions: Overall, the ddPCR assay demonstrated acceptable efficiency in detection of P. knowlesi and P. vivax, and was more sensitive than nested PCR in detecting mixed infections. However, the duplex ddPCR assay still needs optimization to improve the assay's clinical sensitivity and specificity.

The quantification of pathogens is important for assessing water safety and preventing disease outbreaks. Culture-independent approaches, such as quantitative PCR (qPCR) and digital PCR (dPCR), are useful techniques for quantifying pathogens in water samples. However, since pathogens are usually present at low concentrations in water, it is necessary to concentrate microbial cells before extracting their DNA. Many existing microbial concentration methods are inefficient or take a long time to perform. In this study, we applied a coagulation and foam separation method to concentrate environmental water samples of between 1000 and 5000 mL to 100 μL of DNA (i.e., a 1-5 × 10 4 -fold concentration). The concentration process took <1 h. The DNA samples were then used to quantify various target pathogens using dPCR. One gene, the Shiga toxin gene (stx 2 ) of Shiga toxin-producing Escherichia coli, was detected at 32 copies/100 mL in a river water sample. The coagulation and foam concentration method followed by dPCR reported herein is a fast, sensitive, and reliable method to quantify pathogen genes in environmental water samples., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

A CRISPR/Cas12a based portable biosensor (Cas12a-PB) was developed to simultaneously visually detect CaMV35S promoter and Lectin gene from genetically modified (GM) soybean powders (Roundup Ready @ ). The Cas12a-PB, mainly made of polymethylmethacrylate (PMMA) and PMMA tape, has a connection structure, three channels and three detection chambers. The CRISPR/Cas12a detection reagents were preloaded in detection chambers and the reaction tube was connected to the connection structure by screw threads. After amplification, the amplicons were gone into three detection chambers by swinging the Cas12a-PB to conduct dual detection. Positive samples would produce green fluorescence while negative samples were black under the irradiation of 490 nm LED light. In this study, the Cas12a-PB successively combined with ordinary PCR, rapid PCR and loop-mediated isothermal amplification (LAMP) to achieve dual detection, which made detection process more convenient and portable. As low as 0.1% transgenic ingredients in soybean powders could be detected and the specificity of Cas12a-PB was confirmed with GM maize powders (MON810, GA21), GM soybean powders (DP305423), non-GM peanut and rice as targets. In the end, an amplification chamber combining with Cas12a-PB on a PMMA chip was further designed to eliminate the use of reaction tube and mineral oil, which made operation simpler. The established Cas12a-PB would provide a new reliable solution for multiple targets detection in clinic diagnostics, food safety, etc., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A fundamental goal in microbiome studies is determining which microbes affect host physiology. Standard methods for determining changes in microbial taxa measure relative, rather than absolute abundances. Moreover, studies often analyze only stool, despite microbial diversity differing substantially among gastrointestinal (GI) locations. Here, we develop a quantitative framework to measure absolute abundances of individual bacterial taxa by combining the precision of digital PCR with the high-throughput nature of 16S rRNA gene amplicon sequencing. In a murine ketogenic-diet study, we compare microbial loads in lumenal and mucosal samples along the GI tract. Quantitative measurements of absolute (but not relative) abundances reveal decreases in total microbial loads on the ketogenic diet and enable us to determine the differential effects of diet on each taxon in stool and small-intestine mucosa samples. This rigorous quantitative microbial analysis framework, appropriate for diverse GI locations enables mapping microbial biogeography of the mammalian GI tract and more accurate analyses of changes in microbial taxa in microbiome studies.

The "sample-to-answer" integration and automation of circulating tumor DNA (ctDNA)-based liquid biopsy using digital PCR (dPCR) has been hampered by the complicated operations of liquids with volumes ranging from milliliter samples to nanoliter droplets. On the basis of a "3D extensible" design paradigm proposed previously, an integrated droplet digital PCR (IddPCR) microdevice was successfully developed to automate the entire process of liquid biopsy, from the extraction of ctDNA in 2 mL of plasma using magnetic beads to the generation, amplification, and screening of over 30 000 droplets for detection. A series of reagent mixing structures, including macro-, meso-, and micromixers, was designed to enable efficient reagent handling and mixing at different volume scales. The volume thresholds of the microscale and macroscale in the IddPCR device were calculated to be 40 and 100 μL, respectively, based on the fluid dynamics and sizes of the device structures, so that different mixers can be selected according to the reagent volumes. The DNA extraction efficiency obtained on the device was determined to be ∼60%, and the on-chip ddPCR demonstrated a high correlation with an R 2 of 0.9986 between the readouts and the estimations by a Poisson distribution. Finally, the IddPCR microdevice was able to detect rare tumor mutations (T790M) with an occurring frequency as low as ∼1% from 2 mL of human plasma in a "sample-to-answer" manner. This work offers a feasible solution for the automation of liquid biopsy and paves the way for its broad applications in clinics.

We report the development of a versatile system based on the oscillating-flow methodology in a thermal gradient system for nucleic acid analysis. Analysis of DNA and RNA samples were performed in the device, without additional temperature control and complexity. The technique reported in this study eliminates the need for predetermined fluidic channels for thermocycles, and complexity involved with additional incubation steps required for RNA amplification. A microfluidic device was fabricated using rapid prototyping by simply sandwiching dual side adhesive Kapton tape and a polydimethylsiloxane spacer between glass microscope slides. Amplification of the 181-bp segment of a viral phage DNA (ΦX174) and B2M gene in human RNA samples was demonstrated using the system. The developed system enables simultaneous acquisition of amplification and melt curves, eliminating the need for postprocessing. A direct comparison between the oscillating-flow system and a commercial real-time polymerase chain reaction (PCR) instrument showed complete agreement in PCR data and improved sample-to-result time by eliminating an additional 30 min melt curve step required in commercial PCR systems., (© 2020 Wiley Periodicals, Inc.)

Polymerase chain reaction (PCR) is an extremely important tool for molecular diagnosis, as it can specifically amplify nucleic acid templates for sensitive detection. As another division of PCR, free convective PCR was invented in 2001, which can be performed in a capillary tube pseudo-isothermally within a significantly short time. Convective PCR thermal cycling is implemented by inducing thermal convection inside the capillary tube, which stratifies the reaction into spatially separate and stable melting, annealing, and extension zones created by the temperature gradient. Convective PCR is a promising tool that can be used for nucleic acid diagnosis as a point-of-care test (POCT) due to the significantly simplified heating strategy, reduced cost, and shortened detection time without sacrificing sensitivity and accuracy. Here, we review the history of free convective PCR from its invention to development and its commercial applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We present a facile sample partitioning method to enable rapid and low-cost digital PCR (dPCR) assays. By subdividing a high percentage of the sample volume into a large number of equal volume compartments with a self-digitization (SD) chip, this method can achieve a low-waste and high-order sample discretization in a matter of minutes. The SD chip contains a set of parallel microfluidic channels used for sample delivery, and each channel is connected with two rows of cylindrical wells to hold the discretized sample. By utilizing a degassed PDMS sealing slab as a detachable vacuum pumping source, the SD chip automatically generate large arrays of small sample volumes without requirement of external pumping and valving components. Unlike most microfluidic chamber-based methods for sample discretization, our detachable SD chip allows for discretizing sample with air flushing, then peeling off the cover PDMS slab and sealing the digitized samples with oil layer. Due to obviation of time-consuming oil flushing, such microfluidic device can achieve much faster digitization of sample volumes. Furthermore, this digitization chip can partition more than 90% of a sample volume, which is important for the applications where the amount of material available is small. We also demonstrated the utility of the proposed SD chip by applying it to a dPCR assay., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

As biodiversity loss continues to accelerate, there is a critical need for education and biomonitoring across the globe. Portable technologies allow for in situ molecular biodiversity monitoring that has been historically out of reach for many researchers in habitat nations. In the realm of education, portable tools such as DNA sequencers facilitate in situ hands-on training in real-time sequencing and interpretation techniques. Here, we provide step-by-step protocols as a blueprint for a terrestrial conservation genetics field training program that uses low-cost, portable devices to conduct genomics-based training directly in biodiverse habitat countries., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: There is an ongoing collaboration between Field Projects International and Inkaterra Hotels to repeat a genomics-based field conservation program again in Peru. In this case, the program fees will cover airfare and room/board for any instructors, including the authors, and possibly provide minimal compensation if funds permit.

Chip-based dPCR (cdPCR) with a physical boundary between micro-units allows for high parallelism, robustness and sensitivity. However, cross-contamination between micro-units is still a problem that affects the accuracy of results. To overcome this problem, we introduced a heterogeneous modification strategy by microcontact printing to prepare a through-hole microwell chip (TMC) with a hydrophobic exterior surface and hydrophilic interior surface. The modified TMC can reduce cross-contamination (sample residual rate (SRR) of (4.9 ± 1.5)%) by an efficient partitioning yield (unit filling rate (UFR) of (91.1 ± 2.2)%). The sample-residual properties of modified TMCs could be tuned by the reaction conditions. As the contact time increased, the surface CA of the TMC increased, which caused decreases of the SRR and UFR. However, prolonging the contact time to 25 s would cause a sharp reduction of the UFR. The modified TMCs with high UFRs were used for further dPCR studies. The fluorescence images of dPCR chips were collected by fluorescence microscopy and a self-developed optical system, followed by image processing and data statistics to obtain quantitative results. The copy number variation results of the surface hydrophobic TMC was closer to the true value compared to that of the hydrophilic TMC. The results indicated that the sample residue on the hydrophilic TMC would increase the number of positive points, which would cause false positives and clustering error. The absolute quantitative results of gradient dilution plasmid DNA of JAK2 gene using modified TMC also proved that heterogeneous modification made the quantitative results more accurate. The heterogeneous modified TMC is expected to be used for high-throughput, high-sensitivity and high-specificity biological analyses, such as circulating tumor DNA and cell analysis.

Background: Mobile microbiology is an evolving concept that has the potential to reduce morbidity and mortality associated with infectious diseases on a global level. Molecular methods used in the context of mobile microbiology ensure rapid and accurate aetiological diagnostics and allow timely initiation of clinical care. The great majority of published data regarding molecular diagnostics in mobile laboratories have focused on emerging viral infections and using laboratory-developed assays. Use of clinically validated and commercially available molecular diagnostic instruments in routine diagnostics of infectious diseases in mobile laboratories has received only limited attention in the field., Objectives: This review summarizes the suitability of a range of portable diagnostic molecular instruments for application in mobile laboratories by taking into account the instruments' analytical concepts, technical features and environmental requirements, as well as results of major validation studies., Sources: Data on technical features of selected portable instruments were mainly extracted from manufacturers' websites. Information on validation studies of various molecular assays developed for the selected instruments was extracted from peer-reviewed publications searched for through PubMed., Content: Eight portable diagnostic molecular instruments (Alere q, GeneXpert Edge, GeneXpert Omni, Genedrive, PanNAT, Revogene, cobas Liat and ID Now) that are commercially available or in the launching stage are presented and evaluated in the context of the mobile microbiology concept, with particular emphasis on technical features and environmental requirements. Both the cobas Liat and the Alere i assays have been extensively validated in a variety of studies carried out in both adult and paediatric patients from various settings (ranging from primary care to emergency care departments in tertiary centres). Most studies showed comparable performance of cobas Liat and Alere i molecular assays with the standard-of-care in vitro diagnostics molecular assays routinely performed in dedicated/centralized molecular diagnostics laboratories. In addition, acceptable performance of Alere q and Genedrive instruments has been shown in implementations studies for early infant diagnosis of children born to human immunodeficiency virus-positive mothers and detection of hepatitis C virus RNA, respectively. Additional validation studies on existing (GeneXpert Edge, PanNAT, Revogene) and emerging (GeneXpert Omni) technologies are warranted., Implications: Several portable molecular diagnostic platforms reviewed are suitable for mobile microbiology applications. Further development in this field should be directed toward providing a broader range of assays per instrument, multiplexing, reducing the frequency of invalid results, and price cutting., (Copyright © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

In this work we show how DNA microarrays can be produced batch wise on standard microscope slides in a fast, easy, reliable and cost-efficient way. Contrary to classical microarray generation, the microarrays are generated via digital solid phase PCR. We have developed a cavity-chip system made of a PDMS/aluminum composite which allows such a solid phase PCR in a scalable and easy to handle manner. For the proof of concept, a DNA pool composed of two different DNA species was used to show that digital PCR is possible in our chips. In addition, we demonstrate that DNA microarray generation can be realized with different laboratory equipment (slide cycler, manually in water baths and with an automated cartridge system). We generated multiple microarrays and analyzed over 13,000 different monoclonal DNA spots to show that there is no significant difference between the used equipment. To show the scalability of our system we also varied the size and number of the cavities located in the array region up to more than 30,000 cavities with a volume of less than 60 pL per cavity. With this method, we present a revolutionary tool for novel DNA microarrays. Together with new established label-free measurement systems, our technology has the potential to give DNA microarray applications a new boost.

Pathogenic helminth infections are responsible for severe health problems and economic losses worldwide. Timely and accurate diagnosis of helminth infections is critical for adopting suitable strategies for pathogen control. Here, we review recent advances in nucleic acid-based diagnostic methods, including polymerase chain reaction, quantitative qPCR, loop-mediated isothermal amplification and recombinase polymerase amplification, and discuss their advantages and disadvantages for diagnosing helminth infections. In addition, we highlight recent advances in biosensors for the detection of nucleic acid biomarkers that can potentially be used for the diagnosis of helminth infection.

Emerging molecular diagnosis requires ultrafast polymerase chain reaction (PCR) on chip for rapid precise detection of infectious diseases in the point-of-care test. Here, we report nanoplasmonic on-chip PCR for rapid precision molecular diagnostics. The nanoplasmonic pillar arrays (NPA) comprise gold nanoislands on the top and sidewall of large-scale glass nanopillar arrays. The nanoplasmonic pillars enhance light absorption of a white light-emitting diode (LED) over the whole visible range due to strong electromagnetic hotspots between the nanoislands. As a result, they effectively induce photothermal heating for ultrafast PCR thermal cycling. The temperature profile of NPA exhibits 30 cycles between 98 and 60 °C for a total of 3 min and 30 s during the cyclic excitation of white LED light. The experimental results also demonstrate the rapid DNA amplification of both 0.1 ng μL -1 of λ-DNA in 20 thermal cycles and 0.1 ng μL -1 of complementary DNA of Middle East respiratory syndrome coronavirus in 30 thermal cycles using a conventional PCR volume of 15 μL. This nanoplasmonic PCR technique provides a new opportunity for rapid precision molecular diagnostics.

Polymerase chain reaction (PCR) is commonly used for the analysis of nucleic acids in a variety of applications including clinical. There is, however, a need for a low cost portable PCR device that allows rapid identification of pathogenic bacteria. We report a shunting PCR microfluidic device comprising: polycarbonate microfluidic PCR chip; shunting thermal cycler and fluorescence detector. The microfluidic PCR chip - fabricated using micro-milling and thermal fusion bonding for sealing of the cover - was shunted between three double side temperature zones for thermal cycling. Rapid amplification was observed with heating and cooling rates of 1.8 °C/s and 2 °C/s respectively. Lock-in photodetector for fluorescence detection of the microfluidic PCR chip achieved at 95% confidence an LOD of 75pM FITC and 0.7 ng μl -1 of dsDNA using a QuantiFluor assay kit. The device was validated using universal primers - based on chromosomal DNA extracted from non-pathogenic K-12 subtype of Escherichia coli (E. coli) - for amplification of fragments of 250, 552 and 1500 bp. PCR amplification was demonstrated, with annealing temperatures ranging between 54 °C and 68 °C, and confirmed using gel electrophoresis. The developed shunting PCR microfluidic device will allow for low cost and portable nucleic acid amplification for the detection of infectious diseases., (Copyright © 2019 Elsevier B.V. All rights reserved.)

To date, studies on the application of loop-mediated isothermal amplification (LAMP) in the detection of genetically modified organisms (GMOs) are stably increasing and demonstrates LAMP is a potential and promising method for on spot identification of GMOs. However, little information is known for detection of GM potato events by LAMP. In this report, we developed an optimized and visual LAMP assay with high specificity and sensitivity to rapidly amplify genomic DNA of potato EH92-527-1 within 45 min. The limit of detection of LAMP in our study is 10-fold higher than the conventional PCR. Furthermore, LAMP products can be directly observed via naked eyes by addition of SYBR Green I without gel electrophoresis analysis and PCR-based equipment. Therefore, the LAMP assay developed in this paper provides an efficient, convenient and cost-effective tool for the detection of GM potato EH92-527-1.

The great promise of digital PCR is the potential for unparalleled precision enabling accurate measurements for detection and quantification of genetic material. This chapter walks the reader through the fundamentals of digital PCR technology including digital PCR modeling using Poisson statistics. It describes a highly successful implementation of digital PCR technology using the chip-based nanofluidic Applied Biosystems™ QuantStudio™ 3D digital PCR system. It reviews the large number of applications where digital PCR is poised to make significant impacts. These include applications where detection of rare genetic targets is prioritized such as liquid biopsy, rare mutation detection, confirmation of NGS variant detection, detection of fusion transcripts, detection of chimerism and GMO detection and monitoring. These further include applications where accurate quantification of genetic targets is prioritized such as generation of references and standards, copy number variation, and NGS Library quantification.

Tumor DNA sequencing results can have important clinical implications. However, its use is often limited by low DNA input, owing to small tumor biopsy size. To help overcome this limitation we have developed a simple improvement to a commonly used next-generation sequencing (NGS) capture-based library preparation method using formalin-fixed paraffin-embedded-derived tumor DNA. By using on-bead PCR for pre-capture library generation we show that library yields are dramatically increased, resulting in decreased sample failure rates. Improved yields allowed for a reduction in PCR cycles, which translated into improved sequencing parameters without affecting variant calling. This methodology should be applicable to any NGS system in which input DNA is a limiting factor.

Highly-sensitive and contamination-free droplet digital PCR (ddPCR) is an enabling technology and widely needed for accurate quantification of nucleic acid in clinical applications. In this paper, a novel droplet reader was developed by combining a "quasi" confocal laser-induced fluorescence (LIF) cytometry with a delicate microfluidic chip design. The droplets with a size of 90 μm was illuminated at an out-of-focus position by two aligned laser beams to generate maximum fluorescent signal. Additionally, the lateral offset position of the microfluidic chip should be precisely tuned so that the bandwidth of the FAM and VIC channels were configured at the matching sizes. Then, PMT gain voltages and pneumatic pressures were optimized for better droplet detection efficiencies. An aerosol adsorption experiment was performed to demonstrate that there was no aerosol contamination, and detected copy numbers of both mutants and wild types scaled linearly with the expected input copy numbers (r 2 >0.998) with a LoB of 0.0 copies and LoD of 3.0 copies. The results demonstrated that this droplet reader with the delicate chip is a convenient, highly-sensitive and contamination-free to detect fluorescence signals inside droplets after ddPCR, which is highly promising for broad applications of ddPCR in clinical diagnosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)