Portable Nucleic Acid Research Articles

LAMP combined with Pyrococcus furiosus Argonaute for the ultrasensitive and highly specific point-of-care test platform for Listeria monocytogenes detection

Listeria monocytogenes is an important foodborne pathogen that causes serious infectious diseases in humans and animals. Rapid, accurate, and sensitive detection in the field is critical for the timely implementation of control measures and ensuring food safety. The study established a method that combines loop-mediated isothermal amplification (LAMP) technology and Pyrococcus furiosus Argonaute (PfAgo) reporter system, which can detect L. monocytogenes hly gene levels as low as 1.8 × 101 copies. To overcome the demands on instrumentation and equipment, lateral flow strip (LFS) technology was combined for the first time in a LAMP-PfAgo-based system to detect L. monocytogenes. The method was highly sensitive, with a limit of detection of 1.8 × 102copies, and specificity results showed no cross-reactivity with other pathogens. The performance of the method was comparable to that of real-time fluorescence quantitative PCR (qPCR) in the detection of 18 food origin samples. Moreover, the LAMP-PfAgo reaction was equipped a low-cost thin metal bath as a simple amplification device, allowing it to detect L. monocytogenes samples in shorter time, highlighting its potential as a portable, rapid, accurate, and visual nucleic acid assay. The method can facilitate the early detection of pathogenic infections and promoting food safety detection technologies.

Navigating the CRISPR/Cas Landscape for Enhanced Diagnosis and Treatment of Wilson's Disease.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system continues to evolve, thereby enabling more precise detection and repair of mutagenesis. The development of CRISPR/Cas-based diagnosis holds promise for high-throughput, cost-effective, and portable nucleic acid screening and genetic disease diagnosis. In addition, advancements in transportation strategies such as adeno-associated virus (AAV), lentiviral vectors, nanoparticles, and virus-like vectors (VLPs) offer synergistic insights for gene therapeutics in vivo. Wilson's disease (WD), a copper metabolism disorder, is primarily caused by mutations in the ATPase copper transporting beta (ATP7B) gene. The condition is associated with the accumulation of copper in the body, leading to irreversible damage to various organs, including the liver, nervous system, kidneys, and eyes. However, the heterogeneous nature and individualized presentation of physical and neurological symptoms in WD patients pose significant challenges to accurate diagnosis. Furthermore, patients must consume copper-chelating medication throughout their lifetime. Herein, we provide a detailed description of WD and review the application of novel CRISPR-based strategies for its diagnosis and treatment, along with the challenges that need to be overcome.

A phosphorylated guanidine chitosan and UiO-66-NH2 modified magnetic nanoparticle platform for enrichment and detection of multiple bacteria

Wastewater-based epidemiology (WBE) is a powerful tool for early warning of infectious disease outbreaks. Hence, a rapid and portable pathogen monitoring system is urgent needed for on-site detection. In this work, we first reported synthesis of an artificial modulated wide-spectrum bacteria capture nanoparticle (Arg-CSP@UiO@Fe3O4). Arginine-modified phosphorylated chitosan (Arg-CSP) coating could provide strongly positive charged guanidinium group for pathogen interaction by electrostatic attraction, and UiO-66-NH2 layer could help Arg-CSP graft onto Fe3O4 magnetic particles. The capture efficiency of Arg-CSP@UiO@Fe3O4 reached 92.2 % and 97.3 % for Escherichia coli (E.coli) and Staphylococcus epidermidis (S.epidermidis)within 40 min, in 10 mL sample. To prevent pathogen degradation in sewage, a portable nucleic acid extraction-free method was also developed. UiO-66-NH2 could disintegrate in buffer with high concentration of PO43− for bacterium desorption, and then nucleic acid of the bacteria was released by heating. The DNA template concentration obtained by this method was 779.28 times higher than that of the direct thermal lysis product and 8.63 times higher than that of the commercial kit. Afterwards, multiple detection of bacteria was realized by loop-mediated isothermal amplification (LAMP). Artificial regulated pathogen desorption could prevent non-specific adsorption of nucleic acid by nanoparticles. The detection limit of Arg-CSP@UiO@Fe3O4-LAMP method was 80 cfu/mL for E.coli and 300 cfu/mL for S.epidermidis. The accuracy and reliability of the method was validated by spiked sewage samples. In conclusion, this bio-monitoring system was able to detect multiple bacteria in environment conveniently and have good potential to become an alternative solution for rapid on-site pathogen detection.

Generalized predictive analysis of reactions in paper devices via graph neural networks

Microfluidic technology facilitates high-throughput generation of time series data for biological and medical studies. Deep learning enables accurate, predictive analysis and proactive decision-making based on autonomous recognition of intricate pattern hidden in series. In this work, we first devised a paper-based microfluidic system for portable nucleic acid amplification test with economic energy consumption. Then, we employed Graph Neural Network (GNN), distinguished by its non-Euclidean data structure tailored for deep learning, with spatio-temporal attention mechanism to perform near-sensor predictive analysis of the on-chip reaction. Our findings demonstrated that the novel GNN model can provide accurate predictions of positive outcomes at the early stages of the reaction using less than one-third of the total reaction time. Then, the deep learning model trained by on-chip data was subsequently applied to more than 900 clinical plots. Generalization of the GNN model was successfully validated across different detection methods, diverse types of datasets and time series with variable length. Accuracy, sensitivity and specificity of the predictive approach were 96.5 %, 94.3 % and 99.0 % by utilizing the early half of reaction information. Finally, we compared the GNN model with various deep learning models. Despite differences in the prediction of negative samples among various models were minute, GNN obviously offered overall superior performance. This work ignites a cutting-edge application of deep learning in point-of-care and near-sensor tests. By harnessing the power of body area networks and edge/fog computing, our approach unlocks promising possibilities in diverse fields like healthcare and instrument science.

A Portable Nucleic Acid Testing Platform with Photosensitization, a Three-Dimensionally Printed Multipiece Chip, and Digital Color Sensing.

Portable nucleic acid testing (NAT) holds great promise for point-of-care disease diagnosis and field-based applications but remains difficult to achieve. Herein, we describe a portable NAT that streamlines loop-mediated isothermal amplification with photosensitization-based color development in a fully sealed 3D-printed multipiece chip. Using a smartphone accessory and an APP, we also introduce a calibration-free quantification approach via digital color sensing and library matching. With these innovative approaches, our detection platform is highly accessible, allowing for rapid and sensitive NAT without requiring sophisticated instruments and well-trained personnel. The field applicability of our NAT platform was demonstrated by detecting tuberculosis infections in clinical sputum samples and food adulteration in commercial salmon meat products.

An Argonaute-mediated bio-barcode bioassay for one-tube and on-site detection of Staphylococcus aureus

Isothermal amplification method has enabled major advancement toward rapid and deployable detection for nucleic acid. While exciting, there are still a series of deficiencies facing their practical implementation, such as carryover cross-contamination, which are major bottlenecks that must be addressed. In addition, there is a lack of high-throughput detection for foodborne pathogens in foods. Here, we reported an Argonaute-mediated bio-barcode bioassay for one-step cleavage, on-site and high-throughput detection of S. aureus (Staphylococcus aureus) through introducing Tag sequence and recombinase aided amplification (RAA). Furthermore, we designed a single vessel via tube-in-tube manner for one-tube contamination-free and portable detection without opening the lid. With this strategy, the same detection limit as fluorescence detection could be obtained, 1 CFU/mL with high specificity for S. aureus. We further evaluated the performance of bio-barcode biosensing strategy using S. aureus-contaminated clinical and food samples. Argonaute-mediated bio-barcode bioassay had the advantages of modular, universal plug-and-play and high-throughput scanning. This work not only satisfied the requirement for convenient detection but also fulfilled the need for portable on-site nucleic acid detection with portability, adaptability, transferability and practicality.

Development of Recombinase Polymerase Amplification-Lateral Flow Dipstick (RPA-LFD) as a Rapid On-Site Detection Technique for Fusarium oxysporum.

Fusarium oxysporum can cause many important plant diseases worldwide, such as crown rot, wilt, and root rot. During the development of strawberry crown rot, this pathogenic fungus spreads from the mother plant to the strawberry seedling through the stolon, with obvious characteristics of latent infection. Therefore, the rapid and timely detection of F. oxysporum can significantly help achieve effective disease management. Here, we present a protocol for the recombinase polymerase amplification- lateral flow dipstick (RPA-LFD) detection technique for the rapid detection of F. oxysporum on strawberry, which only takes half an hour. A significant advantage of our RPA-LFD technique is the elimination of the involvement of professional teams and laboratories, which qualifies it for field detection. We test this protocol directly on plant samples with suspected infection by F. oxysporum in the field and greenhouse. It is worth noting that this protocol can quickly, sensitively, and specifically detect F. oxysporum in soils and plants including strawberry. Key features • This protocol is used to detect whether plants such as strawberry are infected with F. oxysporum. • This protocol has potential for application in portable nucleic acid detection. • It can complete the detection of samples in the field within 30 min.

Light-Driven Photocatalytic-Photothermal Synergetic System for Portable and Sensitive Nucleic Acid Quantification.

Photosensitizers and photothermal agents have attracted increasing attention for in vitro diagnosis, but the combination remains challenging. Herein, a light-driven photocatalytic-photothermal synergetic system integrated microfluidic distance-based analytical device (PCPT-μDAD) for visual, portable, sensitive, and quantitative detection of targets was developed. Target DNA was recognized and initiated the hybridization chain reaction to form a double-stranded DNA/SYBR Green I (dsDNA/SG-I) complex. By applying the photosensitization of the dsDNA/SG-I complex and the photothermal effect of oxidized 3,3',5,5'-tetramethylbenzidine, the target concentration can effectively translate into a visual distance signal readout. Importantly, the light-driven PCPT-μDAD greatly improves the controllability of catalytic reactions and signal amplification efficiency. The light-driven PCPT-μDAD shows a low limit of detection (fM level), good stability, and high reproducibility for nucleic acid detection.

A Portable Nucleic Acid Sensor Based on PCR for Simple, Rapid, and Sensitive Testing of Botrytis cinerea in Ginseng.

Botrytis cinerea is a ubiquitous pathogen that can infect at least 200 dicotyledonous plant species including many agriculturally and economically important crops. In Ginseng, the fungus may cause ginseng gray mold disease, causing great economic losses in the ginseng industry. Therefore, the early detection of B. cinerea in the process of ginseng production is necessary for the disease prevention and control of the pathogen's spread. In this study, a polymerase chain reaction-nucleic acid sensor (PCR-NAS) rapid detection technique was established, and it can be used for field detection of B. cinerea through antipollution design and portable integration. The present study showed that the sensitivity of PCR-NAS technology is 10 times higher than that of traditional PCR-electrophoresis, and there is no need for expensive detection equipment or professional technicians. The detection results of nucleic acid sensors can be read by the naked eye in under 3 min. Meanwhile, the technique has high specificity for the detection of B. cinerea. The testing of 50 field samples showed that the detection results of PCR-NAS were consistent with those of the real-time quantitative PCR (qPCR) method. The PCR-NAS technique established in this study can be used as a novel nucleic acid field detection technique, and it has a potential application in the field detection of B. cinerea to achieve early warning of the pathogen infection.

An ultrafast and portable nucleic acid detection system based on photothermal PCR and real-time fluorescence detection

The nucleic acid detection technology based on polymerase chain reaction (PCR) is widely used in infectious disease detection due to its excellent sensitivity and specificity. However, the drawbacks of this method include bulky instrumentation in centralized laboratories and an assay time of 1–2 h. Here, we show a portable nucleic acid detection system, successfully performing ultrafast thermocycling and real-time fluorescence detection in a glass microtubule. By using plasmonic nanoparticles, the system can achieve 30 thermocycles in ∼ 2.5 min. The nucleic acid detection efficiency of the system is comparable to that of the conventional thermocycler. The system correctly distinguished the positive and negative samples of the clinical hepatitis B virus (HBV). Overall, this study extends PCR technology to point-of-care use and provides ideas for developing new virus detection systems.

Ultrasensitive and Specific Clustered Regularly Interspaced Short Palindromic Repeats Empowered a Plasmonic Fiber Tip System for Amplification-Free Monkeypox Virus Detection and Genotyping.

The urgent necessity for highly sensitive diagnostic tools has been accentuated by the ongoing mpox (monkeypox) virus pandemic due to the complexity in identifying asymptomatic and presymptomatic carriers. Traditional polymerase chain reaction-based tests, despite their effectiveness, are hampered by limited specificity, expensive and bulky equipment, labor-intensive operations, and time-consuming procedures. In this study, we present a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-based diagnostic platform with a surface plasmon resonance-based fiber tip (CRISPR-SPR-FT) biosensor. The compact CRISPR-SPR-FT biosensor, with a 125 μm diameter, offers high stability and portability, enabling exceptional specificity for mpox diagnosis and precise identification of samples with a fatal mutation site (L108F) in the F8L gene. The CRISPR-SPR-FT system can analyze viral double-stranded DNA from mpox virus without amplification in under 1.5 h with a limit of detection below 5 aM in plasmids and about 59.5 copies/μL when in pseudovirus-spiked blood samples. Our CRISPR-SPR-FT biosensor thus offers fast, sensitive, portable, and accurate target nucleic acid sequence detection.

A portable, high-throughput real-time quantitative PCR device for point-of-care testing

Nucleic acids detection has become essential in the identification of many infectious diseases and tumors. Conventional qPCR instruments are not suitable for point-of-care Moreover, current miniaturized nucleic acid detection equipment has limited throughput and multiplex detection capabilities, typically allowing the detection of a limited number of samples. Here, we present an affordable, portable, and high-throughput nucleic acid detection device for point-of-care detection. This portable device is approximately 220 × 165 × 140 mm in size and about 3 kg in weight. It can provide stable and accurate temperature control and analyze two fluorescent signals (FAM and VIC) and run 16 samples simultaneously. As a proof of concept, we used the two purified DNA samples from Bordetella pertussis and Canine parvovirus and the results showed good linearity and coefficient of variation. Moreover, this portable device can detect as low as 10 copies and has good specificity. Therefore, our device can provide advantages in real-time diagnosis of high-throughput nucleic acid detection in the field, especially for resource-limited conditions.

Paper microfluidics with deep learning for portable intelligent nucleic acid amplification tests.

During global outbreaks such as COVID-19, regular nucleic acid amplification tests (NAATs) have posed unprecedented burden on hospital resources. Data of traditional NAATs are manually analyzed post assay. Integration of artificial intelligence (AI) with on-chip assays give rise to novel analytical platforms via data-driven models. Here, we combined paper microfluidics, portable optoelectronic system with deep learning for SARS-CoV-2 detection. The system was quite streamlined with low power dissipation. Pixel by pixel signals reflecting amplification of synthesized SARS-CoV-2 templates (containing ORF1ab, N and E genes) can be real-time processed. Then, the data were synchronously fed to the neural networks for early prediction analysis. Instead of the quantification cycle (Cq) based analytics, reaction dynamics hidden at the early stage of amplification curve were utilized by neural networks for predicting subsequent data. Qualitative and quantitative analysis of the 40-cycle NAATs can be achieved at the end of 22nd cycle, reducing time cost by 45%. In particular, the attention mechanism based deep learning model trained by microfluidics-generated data can be seamlessly adapted to multiple clinical datasets including readouts of SARS-CoV-2 detection. Accuracy, sensitivity and specificity of the prediction can reach up to 98.1%, 97.6% and 98.6%, respectively. The approach can be compatible with the most advanced sensing technologies and AI algorithms to inspire ample innovations in fields of fundamental research and clinical settings.

Functional Nanopores Enabled with DNA.

Membrane-spanning nanopores are used in label-free single-molecule sensing and next-generation portable nucleic acid sequencing, and as powerful research tools in biology, biophysics, and synthetic biology. Naturally occurring protein and peptide pores, as well as synthetic inorganic nanopores, are used in these applications, with their limitations. The structural and functional repertoire of nanopores can be considerably expanded by functionalising existing pores with DNA strands and by creating an entirely new class of nanopores with DNA nanotechnology. This review outlines progress in this area of functional DNA nanopores and outlines developments to open up new applications.

Functional Nanopores Enabled with DNA

AbstractMembrane‐spanning nanopores are used in label‐free single‐molecule sensing and next‐generation portable nucleic acid sequencing, and as powerful research tools in biology, biophysics, and synthetic biology. Naturally occurring protein and peptide pores, as well as synthetic inorganic nanopores, are used in these applications, with their limitations. The structural and functional repertoire of nanopores can be considerably expanded by functionalising existing pores with DNA strands and by creating an entirely new class of nanopores with DNA nanotechnology. This review outlines progress in this area of functional DNA nanopores and outlines developments to open up new applications.

Automated Microfluidic Nucleic Acid Detection Platform-Integrated RPA-T7-Cas13a for Pathogen Diagnosis

There is a growing urgent need for point-of-care testing (POCT) devices that integrate sample pretreatment and nucleic acid detection in a rapid, economical, and non-labor-intensive way. Here, we have developed an automated, portable nucleic acid detection system employing microfluidic chips integrating rotary valve-assisted sample pretreatment and recombinase polymerase amplification (RPA)-T7-Cas13a into one-step nucleic acid detection. The RPA and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a were integrated into a single-chamber reaction. As a validation model, we used this method to detect Group B streptococci (GBS) DNA and achieved a detection sensitivity of 8 copies/reaction, which is 6 times more sensitive than gold-standard polymerase chain reactions (PCRs). Dual specific recognition of RPA with CRISPR/Cas13a makes our method ultraspecific, with correct detection of Group B streptococci from 8 kinds of pathogenic bacteria. For the 16 positive and 24 negative clinical GBS samples, our assay achieved 100% accuracy compared to the PCR technique. The whole procedure can be automatically completed within 30 min, providing a more robust, sensitive, and accurate molecular diagnostic tool for POCT.

Portable Paper-Based Nucleic Acid Enrichment for Field Testing.

Point-of-care testing (POCT) can be the method of choice for detecting infectious pathogens; these pathogens are responsible for not only infectious diseases such as COVID-19, but also for certain types of cancers. For example, infections by human papillomavirus (HPV) or Helicobacter pylori (H. pylori) are the main cause of cervical and stomach cancers, respectively. COVID-19 and many cancers are treatable with early diagnoses using POCT. A variety of nucleic acid testing have been developed for use in resource-limited environments. However, questions like unintegrated nucleic acid extraction, open detection systems increase the risk of cross-contamination, and dependence on expensive equipment and alternating current (AC) power supply, significantly limit the application of POCT, especially for on-site testing. In this paper, a simple portable platform is reported capable of rapid sample-to-answer testing within 30 min based on recombinase polymerase amplification (RPA) at a lower temperature, to detect SARS-CoV-2 virus and H. pylori bacteria with a limit of detection as low as 4 × 102 copies mL-1 . The platform used a battery-powered portable reader for on-chip one-pot amplification and fluorescence detection, and can test for multiple (up to four) infectious pathogens simultaneously. This platform can provide an alternative method for fast and reliable on-site diagnostic testing.

BaseLess: lightweight detection of sequences in raw MinION data.

With its candybar form factor and low initial investment cost, the MinION brought affordable portable nucleic acid analysis within reach. However, translating the electrical signal it outputs into a sequence of bases still requires mid-tier computer hardware, which remains a caveat when aiming for deployment of many devices at once or usage in remote areas. For applications focusing on detection of a target sequence, such as infectious disease monitoring or species identification, the computational cost of analysis may be reduced by directly detecting the target sequence in the electrical signal instead. Here, we present baseLess, a computational tool that enables such target-detection-only analysis. BaseLess makes use of an array of small neural networks, each of which efficiently detects a fixed-size subsequence of the target sequence directly from the electrical signal. We show that baseLess can accurately determine the identity of reads between three closely related fish species and can classify sequences in mixtures of 20 bacterial species, on an inexpensive single-board computer. baseLess and all code used in data preparation and validation are available on Github at https://github.com/cvdelannoy/baseLess, under an MIT license. Used validation data and scripts can be found at https://doi.org/10.4121/20261392, under an MIT license. Supplementary data are available at Bioinformatics Advances online.

A miniaturized and integrated dual-channel fluorescence module for multiplex real-time PCR in the portable nucleic acid detection system.

A portable nucleic acid detection (PNAD) system based on real-time polymerase chain reaction (real-time PCR) has been developed for point-of-care testing (POCT) of infectious disease pathogens. In order to achieve “sample-in, result-out” while keeping the system compact, the hardware system integrates optical, thermal and motion control modules in a limited space for nucleic acid extraction, purification, amplification and detection. Among these hardware modules, the fluorescence module is one of the most important modules, because its performance directly affects the accuracy and sensitivity of the testing results. In this paper, a miniaturized, high-sensitivity and integrated dual-channel fluorescence module have been proposed for the homemade PNAD system. Based on the principle of confocal optical path, two group of excitation-emission optical paths of different wavelengths are integrated in a small space. In terms of circuitry, a current-light dual negative feedback light emitting diode (LED) drive circuit is applied to improve the stability of the excited light source. All optical and electronic components are integrated in a metal box of 55 mm × 45 mm × 15 mm, that helps miniaturize the detection system. Two different modules have been assembled to fit various fluorescent dyes or probes with the set of excitation and emission as follow: module 1#: 470 nm/525 nm, 570 nm/630 nm; module 2#: 520 nm/570 nm, 630 nm/690 nm. Finally, hepatitis B virus (HBV) concentration gradient detection and multiplex detection of different gene targets of SARS-CoV-2 are carried out on the PNAD system equipped with these two fluorescence modules for evaluating their performances. Compared with the commercial real-time PCR instrument, our fluorescence module has good stability and detection sensitivity.

A Portable Continuous-Flow Polymerase Chain Reaction Chip Device Integrated with Arduino Boards for Detecting Colla corii asini.

Food security is a significant issue in modern society. Because morphological characters are not reliable enough to distinguish authentic traditional Chinese medicines, it is essential to establish an effective and applicable method to identify them to protect people’s health. Due to the expensive cost of the manufacturing process and the large volume of the analytical system, the need to build a portable and cheap device is urgent. This work describes the development of a portable nucleic acid amplification device integrated with thermal control and liquid pumping connecting to Arduino boards. We present a novel microfluidic polymerase chain reaction (PCR) chip with symmetric isothermal zones. The total chip volume is small, and only one Arduino board is needed for thermal control. We assemble a miniaturized liquid pump and program an Arduino file to push the sample mixture into the chip to implement the PCR process. In the proposed operation, the Nusselt number of the sample flow is less than one, and the heat transfer is conduction only. Then we can ensure temperature uniformity in specific reaction regions. A Colla corii asini DNA segment of 200 bp is amplified to evaluate the PCR performance under the various operational parameters. The initial concentration for accomplishing the PCR process is at least 20 ng/μL at the flow rate of 0.4 μL/min in the portable continuous flow PCR (CFPCR) device. To our knowledge, our group is the first to introduce Arduino boards into the heat control and sample pumping modules for a CFPCR device.