Isothermal DNA Amplification Research Articles

Trends and Innovations in Biosensors for COVID-19 Mass Testing.

Fast and widespread diagnosis is crucial to fighting against the outbreak of COVID‐19. This work surveys the landscape of available and emerging biosensor technologies for COVID‐19 testing. Molecular diagnostic assays based on quantitative reverse transcription polymerase chain reaction are used in most clinical laboratories. However, the COVID‐19 pandemic has overwhelmed testing capacity and motivated the development of fast point‐of‐care tests and the adoption of isothermal DNA amplification. Antigenic and serological rapid tests based on lateral‐flow immunoassays suffer from low sensitivity. Advanced digital systems enhance performance at the expense of speed and the need for large equipment. Emerging technologies, including CRISPR gene‐editing tools, benefit from high sensitivity and specificity of molecular diagnostics and the easy use of lateral‐flow assays. DNA sequencing and sample pooling strategies are highlighted to bring out the full capacity of the available biosensor technologies and accelerate mass testing.

Electrochemical Detection of Genomic DNA Utilizing Recombinase Polymerase Amplification and Stem-Loop Probe.

Nucleic acid tests integrated into digital point-of-care (POC) diagnostic systems have great potential for the future of health care. However, current methods of DNA amplification and detection require bulky and expensive equipment, many steps, and long process times, which complicate their integration into POC devices. We have combined an isothermal DNA amplification method, recombinase polymerase amplification, with an electrochemical stem-loop (S-L) probe DNA detection technique. By combining these methods, we have created a system that is able to specifically amplify and detect as few as 10 copies/μL Staphylococcus epidermidis DNA with a total time to result of 70−75 min.

Development of a Sensitive and Rapid Recombinase Polymerase Amplification Assay for Detection of Anaplasma phagocytophilum.

Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the obligate intracellular Gram-negative bacterium Anaplasma phagocytophilum The disease often presents with nonspecific symptoms with negative serology during the acute phase. Direct pathogen detection is the best approach for early confirmatory diagnosis. Over the years, PCR-based molecular detection methods have been developed, but optimal sensitivity is not achieved by conventional PCR while real-time PCR requires expensive and sophisticated instruments. To improve the sensitivity and also develop an assay that can be used in resource-limited areas, an isothermal DNA amplification assay based on recombinase polymerase amplification (RPA) was developed. To do this, we identified a 171-bp DNA sequence within multiple paralogous copies of msp2 within the genome of A. phagocytophilum Our novel RPA assay targeting this sequence has an analytical limit of detection of one genome equivalent copy of A. phagocytophilum and can reliably detect 125 bacteria/ml in human blood. A high level of specificity was demonstrated by the absence of nonspecific amplification using genomic DNA from human or DNA from other closely-related pathogenic bacteria, such as Anaplasma platys, Ehrlichia chaffeensis, Orientia tsutsugamushi, and Rickettsia rickettsii, etc. When applied to patient DNA extracted from whole blood, this new RPA assay was able to detect 100% of previously diagnosed A. phagocytophilum cases. The sensitivity and rapidness of this assay represents a major improvement for early diagnosis of A. phagocytophilum in human patients and suggest a role for better surveillance in its reservoirs or vectors, especially in remote regions where resources are limited.

A Dual-Sensing Thermo-Chemical ISFET Array for DNA-Based Diagnostics.

This paper presents a 32×32 ISFET array with in-pixel dual-sensing and programmability targeted for on-chip DNA amplification detection. The pixel architecture provides thermal and chemical sensing by encoding temperature and ion activity in a single output PWM, modulating its frequency and its duty cycle respectively. Each pixel is composed of an ISFET-based differential linear OTA and a 2-stage sawtooth oscillator. The operating point and characteristic response of the pixel can be programmed, enabling trapped charge compensation and enhancing the versatility and adaptability of the architecture. Fabricated in 0.18 μm standard CMOS process, the system demonstrates a quadratic thermal response and a highly linear pH sensitivity, with a trapped charge compensation scheme able to calibrate 99.5% of the pixels in the target range, achieving a homogeneous response across the array. Furthermore, the sensing scheme is robust against process variations and can operate under various supply conditions. Finally, the architecture suitability for on-chip DNA amplification detection is proven by performing Loop-mediated Isothermal Amplification (LAMP) of phage lambda DNA, obtaining a time-to-positive of 7.71 minutes with results comparable to commercial qPCR instruments. This architecture represents the first in-pixel dual thermo-chemical sensing in ISFET arrays for Lab-on-a-Chip diagnostics.

An LED-Driven AuNPs-PDMS Microfluidic Chip and Integrated Device for the Detection of Digital Loop-Mediated Isothermal DNA Amplification.

The sensitive quantification of low-abundance nucleic acids holds importance for a range of clinical applications and biological studies. In this study, we describe a facile microfluidic chip for absolute DNA quantifications based on the digital loop-mediated isothermal amplification (digital LAMP) method. This microfluidic chip integrates a cross-flow channel for droplet generation with a micro-cavity for droplet tiling. DNA templates in the LAMP reagent were divided into ~20,000 water-in-oil droplets at the cross-flow channel. The droplets were then tiled in the micro-cavity for isothermal amplification and fluorescent detection. Different from the existing polydimethylsiloxane (PDMS) microfluidic chips, this study incorporates gold nanoparticles (AuNPs) into PDMS substrate through silica coating and dodecanol modification. The digital LAMP chip prepared by AuNPs-PDMS combines the benefits of the microstructure manufacturing performance of PDMS with the light-to-heat conversion advantages of AuNPs. Upon illumination with a near infrared (NIR) LED, the droplets were stably and efficiently heated by the AuNPs in PDMS. We further introduce an integrated device with a NIR heating unit and a fluorescent detection unit. The system could detect HBV (hepatitis B virus)-DNA at a concentration of 1 × 101 to 1 × 104 copies/μL. The LED-driven digital LAMP chip and the integrated device; therefore, demonstrate high accuracy and excellent performance for the absolute quantification of low-abundance nucleic acids, showing the advantages of integration, miniaturization, cost, and power consumption.

Integrated nucleic acid testing system to enable TB diagnosis in peripheral settings.

To facilitate treatment and limit transmission of tuberculosis (TB), new methods are needed to enable rapid and affordable diagnosis of the disease in high-burden low-resource settings. We have developed a prototype integrated nucleic acid testing device to detect Mycobacterium tuberculosis (M.tb) in sputum. The device consists of a disposable cartridge and compact, inexpensive instrument that automates pathogen lysis, nucleic acid extraction, isothermal DNA amplification and lateral flow detection. A liquefied and disinfected sputum sample is manually injected into the cartridge, and all other steps are automated, with a result provided in <1.5 h. Cell disruption and DNA extraction is executed within a four-port active valve containing a miniature bead blender (based on PureLyse® technology, Claremont BioSolutions LLC). The DNA-containing eluate is combined with dry master-mix reagents and target DNA is isothermally amplified. Amplified master-mix is then pumped into a lateral flow strip chamber for detection. The entire process is performed in a single-use closed-system cartridge to prevent amplicon carryover. For testing of M.tb-spiked sputum the system provided a limit of detection of 5 × 103 colony forming units (CFU) per mL. None of the negative sputum-only controls yielded a false-positive result. Testing of 45 clinical sputum specimens from TB cases and controls relative to a validated manual qPCR-based comparator method revealed a preliminary sensitivity of 90% and specificity of 96%. With further development, the herein described integrated nucleic acid testing device can enable TB diagnosis and treatment initiation in the same clinical encounter in near-patient low-resource settings of high TB burden countries.

Analysis of Dynamics of a Biphasic Isothermal DNA Amplification Reaction

Isothermal DNA amplification schemes have broad applications, from molecular detection to DNA circuits. A newly developed amplification scheme, the ultrasensitive DNA amplification reaction (UDAR), shows a biphasic output with high endpoint product levels that indicates an endogenous switch. We recently developed a mathematical model that reproduces the dynamics displayed by one type of UDAR template. In this contribution, we use dynamical systems theory to analyze a sequence of models that highlight the features differentiating UDAR from previously analyzed DNA amplification models. This approach allows us to explain the biphasic features as the interaction of solution trajectories with equilibria whose stability is controlled by reaction mechanisms unique to UDAR. This analysis can assist rational design of more sensitive and specific amplification assays.

Elimination of DNA Multimerization Arising from Isothermal Amplification in the Presence of Bst Exo– DNA Polymerase

In recent years, methods of isothermal amplification of nucleic acids using polymerases with a strand-displacement activity have become widespread for identification of specific nucleotide sequences. Bst exo– polymerase is the most popular of these polymerases, although it is inclined to nonspecific amplification (the so-called multimerization), which leads to the accumulation of by-products constructed of tandem nucleotide repeats. In this study, we evaluated the efficiency of multimerization depending on the reaction conditions and proposed some methods for its elimination. The highest efficiency of multimerization was found in the case of Bst 2.0 polymerase in Isothermal buffer, whereas the Bst-like Gss polymerase provided the formation of multimerization products only in Isothermal buffer and at the latest stages of the reaction. The optimal method for elimination of multimerization was the use of Gss polymerase and Thermopol buffer, or Bst LF polymerase and Isothermal II buffer, or Bst 3.0 polymerase and Thermopol buffer, or Bst 3.0 polymerase in Isothermal buffer and Mn2+ ions as a cofactor. In these cases specific isothermal amplification of the target DNA may take place and provide accurate and reliable results.

A stoichiometric and pseudo kinetic model of loop mediated isothermal amplification

Loop mediated isothermal amplification (LAMP) is one of the most popular isothermal DNA amplification techniques for research and commercial applications, enabling amplification of both DNA and RNA (with the assistance of reverse transcriptase). The LAMP mechanism is powered by strategic primer design and a strand displacement polymerase, generating products that fold over, creating loops. LAMP leads to generation of products of increasing length over time. These products containing multiple loops are conventionally called cauliflower structures. Existing literature on LAMP provides extremely limited understanding of progression of cascades of reactions involved in the reaction and it is believed that cauliflower structures of increasing length constitute a majority of the product formed in LAMP. This study presents a first of its kind stoichiometric and pseudo kinetic model to comprehend LAMP reactions in deeper depth by (i) classifying LAMP reaction products into uniquely identifiable categories, (ii) generating a condensed reaction network to depict millions of interconnected reactions occurring during LAMP, and (iii) elucidating the pathways for amplicon generation. Despite the inherent limitations of conventional stoichiometric modelling for polymerization type reactions (the network rapidly becomes too large and intractable), our model provides new theoretical understanding of the LAMP reaction pathway. The model shows that while longer length products are formed, it is the smaller length recycle amplicons that contribute more towards the exponential increase in the amount of double stranded DNA. Prediction of concentration of different types of LAMP amplicons will also contribute substantially towards informing design of probe-based assays.

Recombinase assisted loop-mediated isothermal DNA amplification.

Polymerase chain reaction (PCR) and isothermal amplification methods such as LAMP and RPA are widely used for genetic detection. However, there are some shortcomings of these methods such as dependence on thermocycler instruments for PCR, complexity of primer design, the possibility for nonspecific amplification in LAMP and complexity of components in RPA. We develop a novel isothermal DNA detection system named Recombinase Assisted Loop-mediated Amplification (RALA). Recombinase from Thermus thermophilus (TthRecA) was used to open target double-stranded DNA to initiate loop-mediated amplification under isothermal conditions, which simplified the primer design and circumvented pre-denaturation. A FRET sensor named ProofMan and a proofreading enzyme Pfu were introduced to produce fluorescence signals by cleaving the sensor from the 3' end. Consequently, sequence-specific detection based on the RALA system was achieved, and even a single nucleotide polymorphism (SNP) could be identified. By introducing additional loop primers, the fast RALA version can amplify 102 DNA targets in 30 minutes. In addition to high sensitivity and specificity, the flexibility of choosing different reporting sensors makes this method versatile in either quantitative or qualitative DNA detection.

Rapid diagnostics of genital herpes by loop-mediated isothermal amplification method with fluorescent detection

Introduction. Due to the high clinical significance of herpesvirus diseases, the searching of fast and effective methods for their diagnosis remains relevant.The aim of the study was to evaluate the diagnostic efficiency of the loop-mediated isothermal amplification of DNA with real-time fluorescent detection (RT-LAMP) with SYTO-82 dye on a model of herpes simplex virus (HSV) infection.Materials and methods. A total of 44 urogenital swabs containing type 1 and type 2 HSV DNA and 43 swabs without HSV DNA, including 33 samples containing the DNA of cytomegalovirus, Epstein-Barr virus and herpesvirus type 6, were studied. For RT-LAMP, Bst 2.0 WarmStart DNA polymerase, SYTO-82 dye, LAMP primers were used.Results. The high efficiency of HSV DNA detection in the RT-LAMP reaction with SYTO-82 dye was shown. RT-LAMP in optimal conditions allowed to reduce reaction time for 2-3 times compared to real-time PCR (to 35 minutes). Analytical sensitivity of HSV type 1 and 2 detection in RT-LAMP was 103 copies of DNA/ml. The diagnostic sensitivity and specificity of the RT-LAMP diagnosis of HSV infection were 96% and 100%, respectively.Discussion. RT-LAMP method has a high sensitivity and specificity comparable to RTPCR, while the risk of false positive results obtaining is minimal.Conclusion. Thus, the reaction of RT-LAMP with SYTO-82 dye allows quickly, with high sensitivity and specificity to detect HSV DNA in clinical material and can be considered as a promising point-of-care testing method.

Applying a Linear Amplification Strategy to Recombinase Polymerase Amplification for Uniform DNA Library Amplification.

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification method with broad applications as a point-of-care test and in molecular biology techniques. Currently, most of the applications are focused on target-specific amplification. Because RPA has the advantage of amplifying DNA under isothermal conditions, we utilized RPA as a DNA library amplification tool. In this study, we used a sheared genomic DNA library and an oligonucleotide (oligo) library for the comparison of polymerase chain reaction and RPA. For the sheared DNA library, we observed biased amplification after RPA was conducted. Thus, to amplify the size-variable DNA library uniformly, we introduced a linear amplification strategy with RPA and successfully improved the uniformity. On the other hand, using the same-sized oligo library, we confirmed that RPA amplified this library uniformly without modification of the protocol. These results demonstrate that RPA can be applied not only to amplify a specific target as previously demonstrated but also to amplify a complex DNA library composed of a large number of different DNA molecules.

Sorting out difficulties in immunological diagnosis of neurocysticercosis: Development and assessment of real time loop mediated isothermal amplification of cysticercal DNA in blood

Purpose of the studyAmong various immunological tests available for diagnosis of neurocysticercosis (NCC), only EITB (Electroimmunotransfer blot for detection of anticysticercal antibodies) had gained widespread acceptance. However EITB is not available widely and is costly (Indian rupees 15,000/− approximately). We evaluated utility of Loop mediated isothermal amplification (LAMP) assay for detection of Taenia solium cox1 gene in blood of patients with NCC. Patients and methodsCurrent study included 100 consecutive patients of NCC at a tertiary teaching hospital in Northern India. All the patients underwent detailed history and examinations as well as gadolinium enhanced magnetic resonance imaging of brain. LAMP assay was performed in all the patients. The results were compared with 50 controls. ResultsLAMP detected Taenia Solium cox1 gene in 74% of all blood samples in patients of NCC.T he overall sensitivity of LAMP assay for detection of cox1 gene was 74% in all patients with NCC, 71.8% in patients with intraparenchymal brain cysts only and 86.7% of patients with extraparenchymal brain cysts with or without intraparenchymal brain cysts. The overall specificity of LAMP assay was 90% in all these three subgroups. The positive predictive value of real time LAMP assay was close to 93% for almost all forms of NCC- both solitary and multiple while negative predictive value ranged from 57 to 64%. ConclusionReal time LAMP assay of blood for detection of Taenia solium cox1 gene appears to be a promising toll for diagnosis of NCC.

Development of recombinase polymerase amplification assay for rapid detection of Meloidogyne incognita, M. javanica, M. arenaria, and M. enterolobii

Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification approach that has been used to detect a variety of animal and plant pathogens. However, the RPA assay is rarely used for the molecular diagnosis of plant parasitic nematodes. In this study, we developed RPA assays for the detection of four Meloidogyne spp.; Meloidogyne incognita, M. javanica, M. arenaria, and M. enterolobii. The RPA amplification step could be completed at 38 °C in 20 min without a thermal cycling instrument. The RPA assays were able to distinguish these four Meloidogyne spp. from closely related Meloidogyne species and other plant parasitic nematodes. The detection limits of the RPA assays were 10−2, 10−2, 10−1, and 10−1 dilutions of DNA from a single J2 nematode of M. incognita, M. javanica, M. arenaria, and M. enterolobii, which were less sensitive than polymerase chain reaction (PCR) detection methods. In addition, the RPA assays could detect these four Meloidogyne spp. directly from infested tomato roots. The simplicity, rapidity and practicability all indicated that the RPA assay will be an effective tool for molecular diagnosis of plant parasitic nematodes.

Paper-based Molecular Diagnostics for the Amplification and Detection of Pathogenic Bacteria from Human Whole Blood and Milk Without a Sample Preparation Step

We have developed a novel paper-based molecular pathogen diagnostic method by combining the direct loop-mediated isothermal amplification (Direct LAMP) in a paper reactor and an immunochromatographic strip (ICS) detection without any DNA preparation step. The mineral paper is durable, oil- and tear-resistant, and waterproof, so it is a proper material for point-of-care (POC) testing owing to facile chip fabrication, cost-effectiveness, and disposability. The mineral paper was utilized as a substrate to construct the reactor for gene amplification based on the principle of origami without using an expensive and complicated process. In this novel paper reactor, we performed the Direct LAMP reaction, which is a direct isothermal DNA amplification without any sample preparation step such as cell lysis, DNA extraction and purification for amplifying specific target gene. After the Direct LAMP reaction in a paper reactor, a capillary tube was employed to take an appropriate amount (~ 2 μL) of amplicons in order to transfer the LAMP product to an ICS. The use of the ICS allowed us to perform the simple and rapid colorimetric detection of the resultant amplified target genes by naked eyes, thereby confirming the presence of the pathogen in a sample in a user-friendly way. In this platform, we could simultaneously identify Staphylococcus aureus and Escherichia coli O157:H7 contaminated in human blood or milk. The whole molecular diagnostic process (the Direct LAMP in a mineral paper plus the ICS detection) could be conducted in a paper substrate, so the proposed method would be ideal for pathogen detection in resourcelimited environments.

Single enzyme-based stem-loop and linear primers co-mediated exponential amplification of short gene sequences

Isothermal DNA amplification only using a Bacillus stearothermophilus (Bst) DNA polymerase such as loop-mediated isothermal amplification typically entails multiple target sites for primer design and is thereby not suited for the amplification of short gene sequences, for example, the sequences with size below 200 nucleotides (nt). Here we present SLIMP, a novel single enzyme-based isothermal amplification of short gene sequence mediated by both stem-loop and linear primers. In SLIMP, a pair of stem-loop primers and a pair of linear primers are specifically designed to recognize only two target sites. Linear primers in SLIMP are similar as conventional PCR primers, but stem-loop primers are the modified linear primers through attaching a stem-loop structure at their 5′-ends. Attributed to this unique primer design, three basic reaction modes including linear-priming, single stem-loop-priming, and double stem-loop-priming amplifications co-mediate the SLIMP process under the function of Bst DNA polymerase. As a proof-of-concept assay, a synthetic 80 nt sequence from hepatitis B virus S gene was used as the template to develop SLIMP. On performance, SLIMP detection possesses high sensitivity and specificity, good selectivity, and the potential for analysing real sample. Therefore, SLIMP is expected as a novel alternative to amplify short gene sequences using a single enzyme.

Progress in rolling circle amplification in biological detection

Rolling circle amplification is a rapid, sensitive and isothermal single-stranded DNA amplification technique that can be used with staining or probes to amplify the detection signal. This technology has been widely used in biological detection and other aspects. The present paper introduces how to design rolling circle amplification, summarize its application in the detection of pathogens, nucleic acid tumor markers, proteins, biological small biomolecules, and viruses in recent years and prospects for future development.

Detection and Pulsed-Field Gel Electrophoresis Typing of Listeria monocytogenes Isolates from Milk Vending Machines in Croatia

The aim of the present study was to evaluate the persistence of Listeria monocytogenes in raw milk from vending machines, based on culture and molecular detection of pathogen and Pulsed-Field Gel Electrophoresis typing. From December 2015 to January 2017, a total of 319 milk samples from 36 vending machines were examined for the presence and count of L. monocytogenes by reference methods ISO 11290:1 and ISO 11290:2. Molecular detection of pathogens was performed by loop-mediated isothermal DNA amplification (LAMP) coupled with bioluminescence (Molecular Detection Assay). L. monocytogenes was detected by MDA in 14 milk samples (4.38%) from four farms, compared to eight positive samples (2.5%) retrieved by a reference ISO method. Cultivable L. monocytogenes isolates were subjected to Pulsed-Field Gel Electrophoresis typing and pulsotypes were compared with those obtained during the previous survey in Croatia (2014‒2015). It was found that identical PFGE patterns of L. monocytogenes occur in milk samples of the same producer over a three-year period, indicating the persistence of pathogens in raw milk vending machines. The results obtained support the need for more effective control of milk in the entire food chain.

Establishment of loop-mediated isothermal amplification for rapid and convenient detection of Mycobacterium marinum complex

Mycobacterium marinum is a zoonotic pathogen that can cause dermatological infection mainly from contaminated water or fish. Some well-known genetically similar species and subspecies are M. lifrandii and M. pseudoshottsii from amphibians and fish in aquaculture, and M. ulcerans, a causative agent of a neglected tropical disease (NTD), Buruli ulcer. They are believed to survive in water as their major niche, which might be related to their source of infection, but detailed ecological surveillance of the species complex remains to be done. Herein, we present a new detection system for M. marinum complex based on isothermal DNA amplification that can be conducted conveniently with high sensitivity and specificity. The target was a chromosomal gene, mrsA, including a restriction polymorphism between M. ulcerans (except for the most ancestral subspecies, M. ulcerans subsp. shinshuense) and the other species. The system was able to detect less than 500 fg (approximately 70 copies) of genomic DNA of M. marinum, within 60 min, and caused no amplification from mycobacterial species other than M. marinum complex species. It was also verified that restriction of the amplified DNA fragments was able to discriminate M. ulcerans as expected. This easy, quick, and convenient system is expected to facilitate detection of M. marinum complex from various resources.

Development of a Low-Cost, Wireless Smart Thermostat for Isothermal DNA Amplification in Lab-On-A-Chip Devices.

Nucleic acid amplification tests (NAAT) are widely used for the detection of living organisms, recently applied in Lab-on-a-Chip (LoC) devices to make portable DNA analysis platforms. While portable LoC-NAAT can provide definitive test results on the spot, it requires specialized temperature control equipment. This work focuses on delivering a generalized low-cost, wireless smart thermostat for isothermal NAAT protocols in 2 cm × 3 cm LoC cartridges. We report on the design, prototyping, and evaluation results of our smart thermostat. The thermostat was evaluated by experimental and simulated thermal analysis using 3D printed LoC cartridges, in order to verify its applicability to various isothermal NAAT protocols. Furthermore, it was tested at the boundaries of its operating ambient temperature range as well as its battery life was evaluated. The prototype thermostat was proven functional in 20–30 °C ambient range, capable of maintaining the required reaction temperature of 12 isothermal NAAT protocols with 0.7 °C steady-state error in the worst case.