Nucleic Acid Amplification Reactions Research Articles

Real-Time Monitoring of a Nucleic Acid Amplification Reaction Using a Mass Sensor Based on a Quartz-Crystal Microbalance.

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.

Field-effect transistor biosensor with signal amplification by ternary initiation complexes for detection of wide-range RNA concentration

Electrical detection of RNAs using transistor-based biosensors has attracted attention as a strategy for medical diagnosis and environmental monitoring. Herein, we demonstrated a proof-of-concept for specific, sensitive, and label-free RNA detection using a field-effect transistor (FET) biosensor with signal amplification by ternary initiation complexes (SATIC), which is an isothermal one-step nucleic acid amplification initiated by the combination of target RNA, circular DNA template and DNA primer. The SATIC system-applied FET biosensor specifically and quantitatively detected the target RNA with a single-nucleotide difference via the negative charges derived from the amplification products formed by a nucleic acid amplification reaction with φ29 DNA polymerase on the gate surface. In particular, the control of the amplification time allowed the detection of target RNA molecules over a wide concentration range, resulting in a detection limit of up to 6 copies/μL. Therefore, a transistor-based bioassay using the SATIC system could be useful for simple and sensitive nucleic acid analysis.

Single-Cell Liquid Biopsy of Lung Cancer: Ultra-Simplified Efficient Enrichment of Circulating Tumor Cells and Hand-Held Fluorometer Portable Testing.

Herein, we propose a paper-based laboratory via enzyme-free nucleic acid amplification and nanomaterial-assisted cation exchange reactions (CERs) assisted single-cell-level analysis (PLACS). This method allowed for the rapid detection of mucin 1 and trace circulating tumor cells (CTCs) in the peripheral blood of lung cancer patients. Initially, an independently developed method requiring one centrifuge, two reagents (lymphocyte separation solution and erythrocyte lysate), and a three-step, 45 min sample pretreatment was employed. The core of the detection approach consisted of two competitive selective identifications: copper sulfide nanoparticles (CuS NPs) to C-Ag+-C and Ag+, and dual quantum dots (QDs) to Cu2+ and CuS NPs. To facilitate multimodal point-of-care testing (POCT), we integrated solution visualization, test strip length reading, and a self-developed hand-held fluorometer readout. These methods were detectable down to ag/mL of mucin 1 concentration and the single-cell level. Forty-seven clinical samples were assayed by fluorometer, yielding 94% (30/32) sensitivity and 100% (15/15) specificity with an area under the curve (AUC) of 0.945. Nine and 15 samples were retested by a test strip and hand-held fluorometer, respectively, with an AUC of 0.95. All test results were consistent with the clinical imaging and the folate receptor (FR)-PCR kit findings, supporting its potential in early diagnosis and postoperative monitoring.

Base-Stacking-Driven Catalytic Hairpin Assembly: A Nucleic Acid Amplification Reaction Using Electrode Interface as a "Booster" for SARS-CoV-2 Point-of-Care Testing.

Electrochemical DNA (E-DNA) biosensors based on interface-mediated hybridization reactions are promising for point-of-care testing (POCT). However, the low efficiency of target recycle amplification and the steric hindrance at the electrode interface limit their sensing performance. Herein, we propose a base-stacking-driven catalytic hairpin assembly (BDCHA), a nucleic acid amplification reaction strategy, for POCT. The introduction of the base-stacking effect in this strategy increases the thermodynamic stability of the product, thereby effectively improving the recycling efficiency. Also, it enables the interface-mediated hybridization to maintain stability with even fewer bases in the reaction-binding domain, hence minimizing DNA secondary structure formation or intertwining at the electrode surface and ameliorating the steric hindrance limitation. The introduced base-stacking effect makes the electrode serve as a "booster" by integrating the advantages of homogeneous and heterogeneous reactions, giving BDCHA an increased reaction rate of about 20-fold, compared to the conventional catalytic hairpin assembly. As a proof of concept, our BDCHA was applied in constructing a portable E-DNA biosensor for the detection of a SARS-CoV-2 N gene sequence fragment. A simple 30 min one-pot incubation is required, and the results can be readily read on a smartphone, making it portable and user-friendly for POCT.

Rapid and Ultrasensitive Detection of Plasmodium spp. Parasites via the RPA-CRISPR/Cas12a Platform.

Microscopic examination of thick and thin blood smears stained with Giemsa dye is considered the primary diagnostic tool for the confirmation and management of suspected clinical malaria. However, detecting gametocytes is relatively insensitive, particularly in asymptomatic individuals with low-density Plasmodium infections. To complement existing diagnostic methods, a rapid and ultrasensitive point-of-care testing (POCT) platform for malaria detection is urgently needed and necessary. A platform based on recombinase polymerase amplification (RPA) followed by CRISPR/Cas12a (referred to as RPA-CRISPR/Cas12a) was developed and optimized for the determination of Plasmodium spp. parasites, particularly Plasmodium falciparum, using a fluorescence-based assay (FBDA), lateral flow test strips (LFTS), or naked eye observation (NEO). Then, the established platform was assessed with clinical malaria isolates. Under optimal conditions, the detection threshold was 1 copy/μL for the plasmid, and the limit of detection was 3.11-7.27 parasites/μL for dried blood spots. There was no cross-reactivity against blood-borne pathogens. For the accuracies of RPA-CRISPR/Cas12a, Plasmodium spp. and P. falciparum testing were 98.68 and 94.74%, respectively. The method was consistent with nested PCR results and superior to the qPCR results. RPA-CRISPR/Cas12a is a rapid, ultrasensitive, and reliable platform for malaria diagnosis. The platform requires no or minimal instrumentation for nucleic acid amplification reactions and can be read with the naked eye. Compared with similar diagnostic methods, this platform improves the reaction speed while reducing detection requirements. Therefore, this platform has the potential to become a true POCT for malaria parasites.

Parylene C as a Multipurpose Material for Electronics and Microfluidics.

Poly(p-xylylene) derivatives, widely known as Parylenes, have been considerably adopted by the scientific community for several applications, ranging from simple passive coatings to active device components. Here, we explore the thermal, structural, and electrical properties of Parylene C, and further present a variety of electronic devices featuring this polymer: transistors, capacitors, and digital microfluidic (DMF) devices. We evaluate transistors produced with Parylene C as a dielectric, substrate, and encapsulation layer, either semitransparent or fully transparent. Such transistors exhibit steep transfer curves and subthreshold slopes of 0.26 V/dec, negligible gate leak currents, and fair mobilities. Furthermore, we characterize MIM (metal-insulator-metal) structures with Parylene C as a dielectric and demonstrate the functionality of the polymer deposited in single and double layers under temperature and AC signal stimuli, mimicking the DMF stimuli. Applying temperature generally leads to a decrease in the capacitance of the dielectric layer, whereas applying an AC signal leads to an increase in said capacitance for double-layered Parylene C only. By applying the two stimuli, the capacitance seems to suffer from a balanced influence of both the separated stimuli. Lastly, we demonstrate that DMF devices with double-layered Parylene C allow for faster droplet motion and enable long nucleic acid amplification reactions.

Fluidic Spatial-Confinement Scaffold Affords a Multicomponent DNA Reaction with Improved Efficiency and Accelerated Kinetics.

Enzyme-free nucleic acid amplification reactions with the capability of signal catalytic amplification have been widely used in biosensors. However, these multicomponent, multistep nucleic acid amplification systems often suffer from low reaction efficiency and kinetics. Herein, inspired by the natural cell membrane system, we utilized the red blood cell membrane as a fluidic spatial-confinement scaffold to construct a novel accelerated reaction platform. By simply modifying with cholesterol, DNA components can be efficiently integrated into the red blood cell membrane through hydrophobic interactions, which greatly increases the local concentration of DNA strands. Moreover, the fluidity of the erythrocyte membrane improves the collision efficiency of DNA components in the amplification system. Based on the increased local concentration and improved collision efficiency, the fluidic spatial-confinement scaffold significantly improved the reaction efficiency and kinetics. Taking catalytic hairpin assembly (CHA) as a model reaction, an RBC-CHA probe based on the erythrocyte membrane platform enables a more sensitive detection of miR-21 with a sensitivity that is 2 orders of magnitude higher than the free CHA probe and a fast reaction rate (about 3.3-fold). The proposed strategy provides a new idea for the construction of a novel spatial-confinement accelerated DNA reaction platform.

Multiplex polymerase spiral reaction for simultaneous detection of Salmonella typhimurium and Staphylococcus aureus

Salmonella typhimurium (S. typhimurium) and Staphylococcus aureus (S. aureus) are common food-borne pahogens that cause food poisoning in humans. In this study, we developed a method for the simultaneous determination of S. typhimurium and S. aureus based on multiplex polymerase spiral reaction (m-PSR) and melting curve analysis. Two pairs of primers were designed specifically to target the conserved invA gene of S. typhimurium and nuc gene of S. aureus, and the nucleic acid amplification reaction was achieved under isothermal conditions in the same reaction tube for 40 min at 61 °C, melting curve analysis of the amplification product was carried out. The distinct mean melting temperature allowed simultaneous differentiation of the two target bacteria in the m-PSR assay. The limit of detection of S. typhimurium and S. aureus that could be detected simultaneously was 4.1 × 10−4 ng genomic DNA and 2 × 101 CFU/mL pure bacterial culture. Based on this method, analysis of artificially contaminated samples showed excellent sensitivity and specificity consistent with those of pure bacterial cultures. This method is rapid, simultaneous and promises to be a useful tool for the detection of food-borne pathogens in the food industry.

Sensitive and selective DNA detecting electrochemical sensor via double cleaving CRISPR Cas12a and dual polymerization on hyperbranched rolling circle amplification

Electrochemical sensors are widely used for nucleic acid detection. However, they exhibit low sensitivity and specificity. To overcome these limitations, DNA amplification method is necessary. In this study, we introduced CRISPR (Clustered regularly interspaced short palindromic repeats) Cas12a-dependent hyperbranched rolling circle amplification (HRCA) into an electrochemical sensor platform. By resolving the existing false-positive issue of HRCA, CRISPR Cas12a determines the real positive amplification that able to enhance its sensitivity for extremely low concentrations of nucleic acids and specificity for single-point mutations. In detail, CRISPR Cas12a, which activates the nucleic acid amplification reaction, was used for both trans and cis cleavage for the first time. Finally, selectively amplified DNA was detected using a screen-printed electrode. Using the change in surface coverage by DNA, the electrochemical sensor detected a decrease in the redox signal. In summary, combining a novel DNA amplification method and electrochemical sensor platform, our proposed method compensates for the shortcomings of existing RCA and hyperbranched RCA, secures a high sensitivity of 10 aM, and overcomes false-positivity problems. Moreover, such creative applications of CRISPR Cas12a may lead to the expansion of its applications to other nucleic acid amplification methods.

A protein enzyme-free strategy for fluorescence detection of single nucleotide polymorphisms using asymmetric MNAzymes

To establish protein enzyme-free and simple approach for sensitive detection of single nucleotide polymorphisms (SNPs), the nucleic acid amplification reactions were developed to reduce the dependence on protein enzymes (polymerase, endonuclease, ligase). These methods, while enabling highly amplified analysis for the short sequences, cannot be generalized to long genomic sequences. Herein, we develop a protein enzyme-free and general SNPs assay based on asymmetric MNAzyme probes. The multi-arm probe (MNAzyme-9M-13) with two asymmetric recognition arms, containing a short (9 nt) and a long (13 nt) arm, is designed to detect EGFR T790 M mutation (MT). Owing to the excellent selectivity of short recognition arm, MNAzyme-9M-13 probe can efficiently avoid interferences from wild-type target (WT) and various single-base mutations. Through a one-pot mixing, MNAzyme-9M-13 probe enables the sensitive detection of MT, without protein enzyme or multi-step operation. The calculated detection limit for MT is 0.59 nM and 0.83%. Moreover, this asymmetric MNAzyme strategy can be applied for SNPs detection in long genomic sequences as well as short microRNAs (miRNAs) only by changing the low-cost unlabeled recognition arms. Therefore, along with simple operation, low-cost, protein enzyme-free and strong versatility, our asymmetric MNAzyme strategy provides a novel solution for SNPs detection and genes analysis.

Electrochemical Detection for Isothermal Loop-Mediated Amplification of Pneumolysin Gene of Streptococcus pneumoniae Based on the Oxidation of Phenol Red Indicator.

A highly sensitive electrochemical methodology for end-point detection of loop-mediated isothermal nucleic acid amplification reactions was developed. It is based on the oxidation process of phenol red (PR), commonly used as a visual indicator. The dependence of its redox process on pH, which changes during amplification, allows performing quantitative measurements. Thus, the change in the oxidation potential of PR during the amplification is used, for the first time, as the analytical signal that correlates with the number of initial DNA copies. As a proof-of-concept, the amplification of the pneumolysin gene from Streptococcus pneumoniae, one of the main pathogens causing community-acquired pneumonia, is performed. Combination of isothermal amplification with electrochemical detection, performed on small-size flexible electrodes, allows easy decentralization. Adaptation to the detection of other pathogens causing infectious diseases would be very useful in the prevention of future epidemics.

Nucleic acid amplification strategies for volume-amplified magnetic nanoparticle detection assay.

Magnetic nanoparticles (MNPs) can be quantified based on their magnetic relaxation properties by volumetric magnetic biosensing strategies, for example, alternating current susceptometry. Volume-amplified magnetic nanoparticle detection assays (VAMNDAs) employ analyte-initiated nucleic acid amplification (NAA) reactions to increase the hydrodynamic size of MNP labels for magnetic sensing, achieving attomolar to picomolar detection limits. VAMNDAs offer rapid and user-friendly analysis of nucleic acid targets but present inherence defects determined by the chosen amplification reactions and sensing principles. In this mini-review, we summarize more than 30 VAMNDA publications and classify their detection models for NAA-induced MNP size increases, highlighting the performances of different linear, cascade, and exponential NAA strategies. For some NAA strategies that have not yet been reported in VAMNDA, we predicted their performances based on the reaction kinetics and feasible detection models. Finally, challenges and perspectives are given, which may hopefully inspire and guide future VAMNDA studies.

SMART-LAMP: A Smartphone-Operated Handheld Device for Real-Time Colorimetric Point-of-Care Diagnosis of Infectious Diseases via Loop-Mediated Isothermal Amplification

Nucleic acid amplification diagnostics offer outstanding features of sensitivity and specificity. However, they still lack speed and robustness, require extensive infrastructure, and are neither affordable nor user-friendly. Thus, they have not been extensively applied in point-of-care diagnostics, particularly in low-resource settings. In this work, we have combined the loop-mediated isothermal amplification (LAMP) technology with a handheld portable device (SMART-LAMP) developed to perform real-time isothermal nucleic acid amplification reactions, based on simple colorimetric measurements, all of which are Bluetooth-controlled by a dedicated smartphone app. We have validated its diagnostic utility regarding different infectious diseases, including Schistosomiasis, Strongyloidiasis, and COVID-19, and analyzed clinical samples from suspected COVID-19 patients. Finally, we have proved that the combination of long-term stabilized LAMP master mixes, stored and transported at room temperature with our developed SMART-LAMP device, provides an improvement towards true point-of-care diagnosis of infectious diseases in settings with limited infrastructure. Our proposal could be easily adapted to the diagnosis of other infectious diseases.

Ligation-free isothermal nucleic acid amplification

In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free nucleic acid amplification reaction. In this reaction, DNA elongation occurs from a forward probe to a phosphorothioated-hairpin probe in the presence of target RNA regardless of ligation. The second DNA elongation then occurs simultaneously at the nick site of the phosphorothioated probe and the self-priming region. Therefore, the binding site of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a is repeatedly amplified, inducing a fluorescence signal in the presence of CRISPR-Cas12a. This ligation-free isothermal gene amplification method enables the detection of target RNA with 49.2 fM sensitivity. Moreover, two types of mRNA detection are feasible, thus, demonstrating the potential of this method for cancer companion diagnostics. Notably, the proposed method also demonstrates efficacy when applied for the detection of mRNA extracted from human cells and tumor-bearing mouse tissue and urine samples. Hence, this newly developed ligation-free isothermal nucleic acid amplification system is expected to be widely used in a variety of gene detection platforms.

Bsu polymerase-mediated fluorescence coding for rapid and sensitive detection of 8-oxo-7,8-dihydroguanine in telomeres of cancer cells

Guanine is the most susceptible to oxidation among all the DNA bases, and 8-oxo-7,8-dihydroguanine (OG) is one of main oxidation products that can occur in any part of chromosomal DNA. OG in the telomere sequence is associated with telomere shortening, cell aging, and dysfunction, and it may induce cancers. The accurate detection of OG in telomeres is important to early clinical diagnosis and molecular research. Herein, we develop a simple and rapid method to sensitively measure 8-oxo-7,8-dihydroguanine (OG) in telomeres of cancer cells by using Bsu polymerase-mediated fluorescence coding. This method is very simple without the requirement for any nucleic acid amplification or specific restriction enzyme recognition reaction, and Bsu polymerase can selectively incorporate Cy5-dATP into the opposite site of OG, endowing this method with good specificity. Moreover, the introduction of single-molecule detection significantly improves the sensitivity. This method can detect OG within 70 min with a limit of detection (LOD) of 2.45 × 10−18 M, and it can detect OG in genomic DNA extracted from H2O2-treated HeLa cells with a LOD of 0.0094 ng, holding great potential in disease-specific gene damage research and early clinic diagnosis.

Usefulness of rapid molecular tests in pediatric respiratory tract infections

ObjectiveTo evaluate the role and the advantages of nasopharyngeal swabs in the detection of Influenza A and B viruses and Respiratory syncytial virus through a rapid test based upon a nucleic acid amplification reaction in order to avoid improper antibiotics use.DesignCase-control retrospective study.SettingEmergency Room of “Sandro Pertini” General Hospital, Rome, Italy.ParticipantsChildren (< 14 years old) who consecutively arrived in the Emergency Department (ED) for respiratory tract infections, without obvious signs of bacterial respiratory tract infections and other comorbidities, in the maximal seasonal incidence period of November-to-March of every year between 2016 and 2020.MethodsMedical records of children included in the study were retrospectively examined. Children were subdivided according to the following intervals: 2016–2017 and 2017–2018 (Group 1), 2018–2019 and 2019–2020 (Group 2). Children in Group 2 undertook a nasopharyngeal swab, while those in Group 1 did not undergo any specific diagnostic test.Primary outcomeAvoidance of improper antibiotics administration.ResultsA total of 386 children were included in the study: 174 in Group 1, 212 in Group 2. The Odd Ratio (OR) of prescribing an antibiotic in the groups of children not being swabbed compared to those of children undertaking a swab was 9.21 (CI95% 5.6–15.2, p < 0.001). The overall percentage of hospitalizations, both in the short observation unit or in the pediatric unit, did not differ between the two groups.ConclusionsNasopharyngeal swabs for the detection of Influenza A and B viruses and Respiratory syncytial virus proved to be a useful means to a correct and timely diagnosis and allowed for a significant reduction in the prescription of antibiotic therapy.Trial registrationRetrospectively registered.

Low-Cost, Open-Source Device for High-Performance Fluorescence Detection of Isothermal Nucleic Acid Amplification Reactions.

The ability to detect SARS-CoV-2 is critical to implementing evidence-based strategies to address the COVID-19 global pandemic. Expanding SARS-CoV-2 diagnostic ability beyond well-equipped laboratories widens the opportunity for surveillance and control efforts. However, such advances are predicated on the availability of rapid, scalable, accessible, yet high-performance diagnostic platforms. Methods to detect viral RNA using reverse transcription loop-mediated isothermal amplification (RT-LAMP) show promise as rapid and field-deployable tests; however, the per-unit costs of the required diagnostic hardware can be a barrier for scaled deployment. Here, we describe a diagnostic hardware configuration for LAMP technology, named the FABL-8, that can be built for approximately US$380 per machine and provide results in under 30 min. Benchmarking showed that FABL-8 has a similar performance to a high-end commercial instrument for detecting fluorescence-based LAMP reactions. Performance testing of the instrument with RNA extracted from a SARS-CoV-2 virus dilution series revealed an analytical detection sensitivity of 50 virus copies per microliter-a detection threshold suitable to detect patient viral load in the first few days following symptom onset. In addition to the detection of SARS-CoV-2, we show that the system can be used to detect the presence of two bacterial pathogens, demonstrating the versatility of the platform for the detection of other pathogens. This cost-effective and scalable hardware alternative allows democratization of the instrumentation required for high-performance molecular diagnostics, such that it could be available to laboratories anywhere-supporting infectious diseases surveillance and research activities in resource-limited settings.

Real-time isothermal nucleic acid amplification detection in resource-limited settings: a description of an open-source miniature fluorimeter

BackgroundThe detection and quantification of nucleic acids traditionally relies on PCR, which requires the use of expensive thermocyclers with integrated fluorescence detection of amplicons. While isothermal nucleic acid amplification technologies eliminate the need for thermocycling, fluorescence-based detection of products is still required for real-time, quantitative readout. Several commercially available isothermal heaters with integrated fluorescence detection exist, but the cost of these devices remains a significant barrier to the use of isothermal nucleic acid amplification in low-resource settings. A low-cost device for nucleic acid amplification detection could be used to contain the spread of the SARS-CoV-2 virus during the current pandemic as well as other disease targets in the future. Increasing COVID-19 testing capacity is particularly relevant when variations of the virus that appear to be more infectious are spreading rapidly and efforts to scale up delivery of COVID-19 vaccines are in the early stages. Here, we describe a modular, low-cost fluorimeter for use in resource-limited settings that lack access to and infrastructure for traditional testing platforms. MethodsWe present a fluorimeter that is constructed from off-the-shelf components enclosed in a compact 3D printed housing that removes the need for specialised optical alignment. The fluorimeter is designed to be placed atop a commercially available heat block holding a PCR-tube and was optimised to detect fluorescein (FITC) dye. The modular design allows it to be easily adapted for use with other dyes commonly used as reporters in nucleic acid amplification reactions. FindingsClinical applicability of the system was demonstrated using the system to measure amplification with two different isothermal amplification technologies with significantly different temperature, volume, and fluorescence requirements: recombinase polymerase amplification (RPA) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). RPA detection was demonstrated with a positive control DNA provided in a commercial kit while RT-LAMP detection of clinically meaningful levels of SARS-CoV-2 RNA was demonstrated in a custom RT-LAMP assay. In a preliminary analysis of nasopharyngeal swab samples from 74 individuals, the custom RT-LAMP assay showed a positive agreement of 92·3% and a negative agreement of 91·4% with the SARS-CoV-2 RT-qPCR test developed by the US Centers for Disease Control and Prevention. InterpretationThis open-source system was designed to improve accessibility to nucleic acid testing with isothermal amplification methods. The system can be easily produced using only a low-end 3D printer. Moreover, the use of single-board computers obviates the need to produce printed circuit boards. At a time when global supply chains are at their most fragile, open-source diagnostic equipment has the potential to reduce pandemic-related health inequities. FundingInternational AIDS vaccine Initiative IAVI and USAID: award AID-OAA-A16-00032.

A review of reaction enhancement strategies for isothermal nucleic acid amplification reactions

Isothermal nucleic acid amplification techniques are used to detect a variety of molecules such as RNAs, DNAs and proteins. When compared to the gold standard polymerase chain reaction, isothermal methods are rapid and simple but often have lower specificity and sensitivity. In this review, we provide a summary of reaction additives and techniques that enhance the sensitivity, specificity, stability, robustness, or speed of isothermal nucleic acid amplification reactions used for sensing applications. We also provide the mechanism and purpose of each enhancement technique, which can provide guidance when selecting reaction additives to optimize novel isothermal amplification systems and assays.

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings.

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.