Search Results (67)

Efficient control measures for respiratory diseases in humans and farm animals require accurate, specific, and rapid diagnostics. Traditional PCR-based molecular diagnostics are restricted to centralized laboratories, which results in significant, potentially catastrophic delays in test results. A case in point is the recent avian flu outbreak, which has culled more than 280 million poultry birds worldwide (over 157 million in the USA alone) since 2022; has spread to other farm animals, such as cattle; has further heightened the risk of a human pandemic; and threatens food security. To enable molecular diagnosis of bird respiratory diseases at the point of need, we employ loop-mediated isothermal amplification (LAMP) in two platforms: (A) portable devices linked to a smartphone and (B) an inexpensive, disposable, electricity-free, instrument-free device with closed-tube, colorimetric detection that can be produced with minimal resources. Smartphone integration offers an unexplored opportunity for spatiotemporal disease mapping, equipping policymakers with critical data for outbreak control. Our assays demonstrated 100% sensitivity and specificity compared to the gold standard, lab-based, quantitative PCR (qPCR). We tested contrived samples of the avian flu H5N1 virus, laryngotracheitis virus (ILTV), and infectious bronchitis virus (IBV) spiked into clinical samples, achieving a detection sensitivity adequate for early infection diagnosis in under 45 min. The test is simple, requires minimal training, and can be performed without refrigeration, making it well-suited for resource-limited settings. Full article

Human papillomavirus (HPV) is the most prevalent sexually transmitted infection worldwide and a leading cause of cervical cancer, accounting for over 300,000 deaths annually, primarily due to high-risk genotypes HPV-16 and HPV-18. Conventional molecular diagnostic methods, such as polymerase chain reaction (PCR), require expensive instrumentation and well-equipped laboratories, which limits their applicability in low-resource or decentralized settings. To address this challenge, the aim of this study was to develop a prototype point-of-care (POC) diagnostic platform based on helicase-dependent amplification (HDA) integrated into a microfluidic device for the specific detection of HPV-16 and HPV-18. The proposed POC platform comprises a disposable poly (methyl methacrylate) (PMMA) microfluidic device, a portable warming mat for isothermal amplification at 65 °C, and a compact electrophoresis chamber for fluorescence-based visualization using SYBR Safe dye, with an approximate total cost of $320 USD. Platform validation was performed on 33 samples, demonstrating amplification of target sequences in less than 60 min with only 20 µL of reaction volume, a limit of detection (LOD) of 15 copies (cp) per reaction, a sensitivity of 95.52%, and a specificity of 100%. This portable and scalable platform constitutes a cost-effective and reliable tool for the detection of HPV, supporting global health initiatives, including those driven by the World Health Organization (WHO), aimed at eliminating cervical cancer as a public health threat, as it can be implemented in decentralized or resource-limited settings. Full article

Infectious diseases poses a growing public health challenge. The COVID-19 pandemic has further emphasized the urgent need for rapid, accessible diagnostics. This study presents the development of an integrated, flexible point-of-care (POC) diagnostic system for the rapid detection of Corynebacterium diphtheriae, the pathogen responsible for diphtheria. The system comprises a microfluidic polymerase chain reaction (micro-PCR) device and an electrochemical DNA biosensor, both fabricated on flexible substrates. The micro-PCR platform offers rapid DNA amplification overcoming the time limitations of conventional thermocyclers. The biosensor utilizes specific molecular recognition and an electrochemical transducer to detect the amplified DNA fragment, providing a clear and direct indication of the pathogen’s presence. The combined system demonstrates the effective amplification and detection of a gene fragment from a toxic strain of C. diphtheriae, chosen due to its increasing incidence. The design leverages lab-on-a-chip (LOC) and microfluidic technologies to minimize reagent use, reduce cost, and support portability. Key challenges in microsystem design—such as flow control, material selection, and reagent compatibility—were addressed through optimized fabrication techniques and system integration. This work highlights the feasibility of using flexible, integrated microfluidic and biosensor platforms for the rapid, on-site detection of infectious agents. The modular and scalable nature of the system suggests potential for adaptation to a wide range of pathogens, supporting broader applications in global health diagnostics. The approach provides a promising foundation for next-generation POC diagnostic tools. Full article

This study presents the MSPAT (Multispectral Soil Plant Analysis Tool), a device designed for assessing leaf nitrogen concentrations in maize crops under field conditions. The MSPAT includes the AS7265x sensor, which has 18 bands and covers the spectrum from 410 to 940 nm. This device was designed to be portable, using the ESP32 microcontroller and incorporating such functionalities as data storage on a MicroSD card, communication with a smartphone via Wi-Fi, and geolocation of acquired data. The MSPAT was evaluated in an experiment conducted at the Federal University of Ceará (UFC), where maize was subjected to different doses of nitrogen fertiliser (0, 60, 90, 120, 150, and 180 kg·ha⁻¹ N). Spectral readings were taken at three phenological stages (V5, V10, and R2) using the MSPAT, an SPAD-502 chlorophyll meter, and a FieldSpec PRO FR3 spectroradiometer. After the optical measurements were taken, the nitrogen concentrations in the leaves were determined in a laboratory by using the Kjeldahl method. The data analysis included the calculation of normalised ratio indices (NRIs) using linear regression and the application of multivariate statistical methods (PLSR and PCR) for predicting leaf nitrogen concentrations (LNCs). The best performance for the MSPAT index (NRI) was obtained using the 900 nm and the 560 nm bands (R² = 0.64) at stage V10. In the validation analysis, the MSPAT presented an R² of 0.79, showing performance superior to that of SPAD-502, which achieved an R² of 0.70. This confirms the greater potential of the MSPAT compared to commercial equipment and makes it possible to obtain results similar to those obtained using the reference spectroradiometer. The PLSR model with data from the FieldSpec 3 provided important validation metrics when using reflectance data with first-derivative transformation (R² = 0.88, RMSE = 1.94 and MAE = 1.28). When using the MSPAT, PLSR (R² = 0.75, RMSE = 2.77 and MAE = 2.26) exhibited values of metrics similar to those for PCR (R² = 0.75, RMSE = 2.78 and MAE = 2.26). This study validates the use of MSPAT as an effective tool for monitoring the nutritional status of maize to optimize the use of nitrogen fertilisers. Full article

Thread-based analytical devices are low-cost, portable, and easy to use, making them ideal for detecting various biomolecules like glucose and DNA with minimal sample requirements, while also offering environmental benefits through their biodegradability. This study explores the potential of zwitterionic poly(sulfobetaine methacrylate) brushes modified cotton thread (PSBMA@threads) as an innovative substitute for DNA solid-phase extraction. The PSBMA polymer brushes were synthesized on cotton threads via surface-initiated atom transfer radical polymerization (SI-ATRP). The usability of the PSBMA@threads for DNA extraction from cell lysates containing cell debris, proteins, and detergents was evaluated. Characterization using SEM, FTIR, and EDS confirmed the successful functionalization with PSBMA polymer brushes. The antifouling properties of PSBMA@threads, including resistance to non-specific protein adsorption and underwater oil repellency, were assessed. The results demonstrated selective DNA capture from protein and lipid-rich lysates. Optimized extraction parameters improved DNA yield, enabling efficient extraction from tumor cells, which successfully underwent PCR amplification. Comparative experiments with commercial silica membrane-based columns revealed that PSBMA@threads exhibited comparable DNA extraction capability. The PSBMA@threads maintained extraction capability after six months of ambient storage, highlighting its stability and cost-effectiveness for nucleic acid isolation in analytical applications. Full article

Multiplex Polymerase Chain Reaction (PCR) has significantly impacted the field of infectious disease diagnostics, offering rapid and precise identification of bacterial and fungal pathogens. Unlike traditional culture methods, which may take days to yield results, multiplex PCR provides diagnostic insights within hours, enabling faster, targeted antimicrobial therapy and reducing the delay in treating critical infections like sepsis. The technique’s high sensitivity and broad pathogen coverage make it ideal for both single and polymicrobial infections, improving outcomes across respiratory, bloodstream, and bacterial/fungal infections. However, multiplex PCR is not without challenges; initial high costs and the need for specialized training can limit its adoption, especially in low-resource settings. This review discusses the clinical advantages and limitations of multiplex PCR, highlighting its influence on diagnostic accuracy, antimicrobial stewardship, and the global fight against antimicrobial resistance (AMR). Furthermore, recent innovations in multiplex PCR, such as digital PCR and portable devices, are explored as potential tools for expanding access to rapid diagnostics worldwide. Full article

Biological warfare agents are infectious microorganisms or toxins capable of harming or killing humans. Francisella tularensis is a potential bioterrorism agent that is highly infectious, even at very low doses. Biosensors for biological warfare agents are simple yet reliable point-of-care analytical tools. Developing highly sensitive, reliable, and cost-effective label-free DNA biosensors poses significant challenges, particularly when utilizing traditional techniques such as fluorescence, electrochemical methods, and others. These challenges arise primarily due to the need for labeling, enzymes, or complex modifications, which can complicate the design and implementation of biosensors. In this study, we fabricated Graphene Quantum dot (GQD)-functionalized biosensors for highly sensitive label-free DNA detection. GQDs were immobilized on the surface of screen-printed gold electrodes via mercaptoacetic acid with a thiol group. The single-stranded DNA (ssDNA) probe was also immobilized on GQDs through strong π−π interactions. The ssDNA probe can hybridize with the ssDNA target and form double-stranded DNA, leading to a decrease in the effect of GQD but a positive shift associated with the increase in DNA concentration. The specificity of the developed system was observed with different microorganism target DNAs and up to three-base mismatches in the target DNA, effectively distinguishing the target DNA. The response time for the target DNA molecule is approximately 1010 s (17 min). Experimental steps were monitored using UV/Vis spectroscopy, Atomic Force Microscopy (AFM), and electrochemical techniques to confirm the successful fabrication of the biosensor. The detection limit can reach 0.1 nM, which is two–five orders of magnitude lower than previously reported methods. The biosensor also exhibits a good linear range from 10⁵ to 0.01 nM and has good specificity. The biosensor’s detection limit (LOD) was evaluated as 0.1 nM from the standard calibration curve, with a correlation coefficient of R² = 0.9712, showing a good linear range and specificity. Here, we demonstrate a cost-effective, GQD-based SPGE/F. tularensis DNA test suitable for portable electrochemical devices. This application provides good perspectives for point-of-care portable electrochemical devices that integrate sample processing and detection into a single cartridge without requiring a PCR before detection. Based on these results, it can be concluded that this is the first enzyme-free electrochemical DNA biosensor developed for the rapid and sensitive detection of F. tularensis, leveraging the nanoenzyme and catalytic properties of GQDs. Full article

The H9 subtype of avian influenza virus (AIV) has been characterized by its rapid spread, wide range of prevalence, and continuous evolution in recent years, leading to an increasing ability for cross-species transmission. This not only severely impacts the economic benefits of the aquaculture industry, but also poses a significant threat to human health. Therefore, developing a rapid and sensitive detection method is crucial for the timely diagnosis and prevention of H9 AIVs. In this study, a real-time fluorescent reverse transcription recombinase-aided isothermal amplification (RT–RAA) technique targeting the hemagglutinin (HA) of H9 AIVs was established. This technique can be used for detection in just 30 min at a constant temperature of 42 °C, and it exhibits good specificity without cross-reactivity with other viruses. Sensitivity tests revealed that the detection limit of RT–RAA was 163 copies per reaction, and the visual detection limit was 1759 copies per reaction at a 95% confidence interval, both of which are capable of detecting low concentrations of standards. Furthermore, RT–RAA was applied to detect 155 clinical samples, and compared to real-time fluorescent quantitative PCR (RT–qPCR), RT–RAA demonstrated high accuracy, with a specificity of 100% and a kappa value of 0.96, indicating good correlation. Additionally, with the assistance of a portable blue imaging device, we can visually observe the amplification products, greatly facilitating rapid detection in resource-limited environments. The RT–RAA detection method developed in this study does not require expensive equipment or highly skilled staff, making it beneficial for the accurate and low-cost detection of H9 AIVs. Full article

As of 31 October 2023, there have been 771,795,258 confirmed cases of COVID-19 globally. Developing simple, portable, and reliable testing devices has become increasingly important. This paper presents a point-of-care testing (POCT) device for COVID-19 based on the dual-excitation fluorescence RT-LAMP method, which is derived from the principles of RT-LAMP-based COVID-19 detection kits available in the market. The key design solutions of the device were simulated and modeled. Key performance metrics such as detection repeatability and linearity were validated. Comparative experiments with the RT-qPCR detection method were conducted to verify the accuracy and reliability of the device. Additionally, the device’s detection sensitivity and accuracy were assessed. Experimental results show that the repeatability coefficient of variation (CV) value is ≤0.09%; the linearity R² for the FAM channel is 0.9977 and that for the HEX channel is 0.9899; it exhibits good anti-interference performance, with negligible cross-channel interference; the temperature stability is ±0.062 °C, the temperature accuracy is less than 0.2 °C, and there is no significant temperature overshoot during the heating process. Compared with the real-time quantitative PCR (RT-qPCR) instrument, the positive agreement rate is 100% and the negative agreement rate is 95.0%. This research provides a foundational basis for the development of equipment for the prevention of infectious diseases and clinical diagnostics. Full article

Timely and accurate detection of viruses is crucial for infection diagnosis and treatment. However, it remains a challenge to develop a portable device that meets the requirement of being portable, powerless, user-friendly, reusable, and low-cost. This work reports a compact ∅30 × 48 mm portable powerless isothermal amplification detection device (material cost ∼$1 USD) relying on LAMP (Loop-Mediated Isothermal Amplification). We have proposed chromatographic-strip-based microporous permeation technology which can precisely control the water flow rate to regulate the exothermic reaction. This powerless heating combined with phase-change materials can maintain a constant temperature between 50 and 70 °C for a duration of up to 49.8 min. Compared with the conventional methods, it avoids the use of an additional insulation layer for heat preservation, greatly reducing the size and cost. We have also deployed a color card and a corresponding algorithm to facilitate color recognition, data analysis, and storage using a mobile phone. The experimental results demonstrate that our device exhibits the same limit of detection (LOD) as the ProFlex PCR for SARS-CoV-2 pseudovirus samples, with that for both being ${10}^3$ copies/μL, verifying its effectiveness and reliability. This work offers a timely, low-cost, and easy way for respiratory infectious disease detection, which could provide support in curbing virus transmission and protecting the health of humans and animals, especially in remote mountainous areas without access to electricity or trained professionals. Full article

Nucleic acid amplification reactions such as polymerase chain reaction (PCR), which uses a DNA polymerase to amplify individual double-stranded DNA fragments, are a useful technique for visualizing the presence of specific genomes. Although the fluorescent labeling method is mainly used with DNA amplification, other detection methods should be considered for further improvements, such as miniaturization and cost reduction, of reaction-monitoring devices. In this study, the quartz-crystal microbalance (QCM) method, which can measure nanogram-order masses, was applied for the real-time detection of DNA fragments in a solution with nucleic acids. This was combined with an isothermal nucleic acid amplification reaction based on the recombinase polymerase amplification (RPA) method, which allowed DNA amplification at a constant temperature. When the DNA amplification reaction was initiated on a QCM sensor plate with an immobilized primer DNA strand, a significant increase in mass was observed compared to when the primer DNA was not immobilized. QCM was shown to be sufficiently sensitive for the in situ detection of amplified DNA fragments. Combining a portable QCM device and RPA offers a sensitive point-of-care method for detecting nucleic acids. Full article

The rapid and sensitive detection of foodborne pathogens is crucial for ensuring food safety. Among virus testing methods, polymerase chain reaction (PCR) has served as the gold-standard technique in most food safety regulation organizations. However, to enhance the speed and efficiency of PCR, novel approaches are continually being explored. In this work, leveraging the photothermal effects and high thermal conductivity of gold nanoparticles, we have significantly improved the heating and cooling rates of thermal cycles, enabling ultra-fast PCR detection. Specifically, we present a pre-degassing multiplex digital PCR chip integrated with gold nanoparticles. We further developed a portable system with a light source for photothermal heating cycling, along with an optoelectronic sensor to analyze PCR amplification products after rapid thermal cycling. As proof of concept, the proposed chip and portable device was applied for the on-site detection of several types of foodborne pathogens, including Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, and Salmonella. The whole system could distinguish those pathogens within 20 min, showing good potential for the rapid detection of multiple types of foodborne pathogens. Full article

Accurate sample heating is vital for nucleic acid extraction and amplification, requiring a sophisticated thermal cycling process in nucleic acid detection. Traditional molecular detection systems with heating capability are bulky, expensive, and primarily designed for lab settings. Consequently, their use is limited where lab systems are unavailable. This study introduces a technique for performing the heating process required in molecular diagnostics applicable for point-of-care testing (POCT), by presenting a method for crafting customized heaters using freely patterned nichrome (NiCr) wire. This technique, fabricating heaters by arranging protrusions on a carbon black-polydimethylsiloxane (PDMS) cast and patterning NiCr wire, utilizes cost-effective materials and is not constrained by shape, thereby enabling customized fabrication in both two-dimensional (2D) and three-dimensional (3D). To illustrate its versatility and practicality, a 2D heater with three temperature zones was developed for a portable device capable of automatic thermocycling for polymerase chain reaction (PCR) to detect Escherichia coli (E. coli) O157:H7 pathogen DNA. Furthermore, the detection of the same pathogen was demonstrated using a customized 3D heater surrounding a microtube for loop-mediated isothermal amplification (LAMP). Successful DNA amplification using the proposed heater suggests that the heating technique introduced in this study can be effectively applied to POCT. Full article

Phalaenopsis orchids are one of the most popular ornamental plants. More than thirty orchid viruses have been reported, and virus-infected Phalaenopsis orchids significantly lose their commercial value. Therefore, the development of improved viral disease detection methods could be useful for quality control in orchid cultivation. In this study, we first utilized the MinION, a portable sequencing device based on Oxford Nanopore Technologies (ONT) to rapidly detect plant viruses in Phalaenopsis orchids. Nanopore sequencing revealed the presence of three plant viruses in Phalaenopsis orchids: odontoglossum ringspot virus, cymbidium mosaic virus, and nerine latent virus (NeLV). Furthermore, for the first time, we detected NeLV infection in Phalaenopsis orchids using nanopore sequencing and developed the reverse transcription–recombinase polymerase amplification (RT-RPA)-CRISPR/Cas12a method for rapid, instrument-flexible, and accurate diagnosis. The developed RT-RPA-CRISPR/Cas12a technique can confirm NeLV infection in less than 20 min and exhibits no cross-reactivity with other viruses. To determine the sensitivity of RT-RPA-CRISPR/Cas12a for NeLV, we compared it with RT-PCR using serially diluted transcripts and found a detection limit of 10 zg/μL, which is approximately 1000-fold more sensitive. Taken together, the ONT platform offers an efficient strategy for monitoring plant viral pathogens, and the RT-RPA-CRISPR/Cas12a method has great potential as a useful tool for the rapid and sensitive diagnosis of NeLV. Full article

Digital droplet PCR (ddPCR) is a powerful amplification technique for absolute quantification of viral nucleic acids. Although commercial ddPCR devices are effective in the lab bench tests, they cannot meet current urgent requirements for on-site and rapid screening for patients. Here, we have developed a portable and fully integrated lab-on-a-disc (LOAD) device for quantitively screening infectious disease agents. Our designed LOAD device has integrated (i) microfluidics chips, (ii) a transparent circulating oil-based heat exchanger, and (iii) an on-disc transmitted-light fluorescent imaging system into one compact and portable box. Thus, droplet generation, PCR thermocycling, and analysis can be achieved in a single LOAD device. This feature is a significant attribute for the current clinical application of disease screening. For this custom-built ddPCR setup, we have first demonstrated the loading and ddPCR amplification ability by using influenza A virus-specific DNA fragments with different concentrations (diluted from the original concentration to 10⁷ times), followed by analyzing the droplets with an external fluorescence microscope as a standard calibration test. The measured DNA concentration is linearly related to the gradient–dilution factor, which validated the precise quantification for the samples. In addition to the calibration tests using DNA fragments, we also employed this ddPCR-LOAD device for clinical samples with different viruses. Infectious samples containing five different viruses, including influenza A virus (IAV), respiratory syncytial virus (RSV), varicella zoster virus (VZV), Zika virus (ZIKV), and adenovirus (ADV), were injected into the device, followed by analyzing the droplets with an external fluorescence microscope with the lowest detected concentration of 20.24 copies/µL. Finally, we demonstrated the proof-of-concept detection of clinical samples of IAV using the on-disc fluorescence imaging system in our fully integrated device, which proves the capability of this device in clinical sample detection. We anticipate that this integrated ddPCR-LOAD device will become a flexible tool for on-site disease detection. Full article