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Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. Considering the onsite use of RPA reagents, lyophilized RPA reagents with long storage stability are highly desired. In this study, as one of the approaches to solve this problem, we attempted to use a thermostable pyruvate kinase (PK). PK gene was isolated from a thermophilic bacterium Thermotoga maritima (Tma -PK). Tma -PK was expressed in Escherichia coli and purified from the cells. Tma -PK exhibited higher thermostability than human PK. The purified Tma -PK preparation was applied to RPA as an ATP-regenerating enzyme. Liquid RPA reagent with Tma -PK exhibited the same performance as that with human PK. Lyophilized RPA reagent with Tma -PK exhibited higher performance than that with human PK. Combined with our previous results of RPA reagents of thermostable Pol from a thermophilic bacterium, Aeribacillus pallidus , the results in this study suggest that thermostable enzymes are preferable to mesophilic ones as a component in lyophilized RPA reagents. [ABSTRACT FROM AUTHOR]

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41°C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. In this study, we attempted to use pyruvate kinase instead of creatine kinase (CK) that has been consistently used as an ATP-regenerating enzyme in RPA. Human pyruvate kinase M1 (PKM) was expressed in Escherichia coli and purified from the cells. RPA with PKM was performed at 41°C with the in vitro synthesized urease subunit β (ureB) DNA from Ureaplasma parvum serovar 3 as a standard DNA. The optimal concentrations of PKM and phosphoenolpyruvate were 20 ng/μL and 10 mM, respectively. The RPA reaction with PKM was more sensitive than that with CK. PKM exhibited higher thermostability than CK, suggesting that the RPA reagents with PKM are preferable to those with CK for onsite use. [ABSTRACT FROM AUTHOR]

Analytical systems based on isothermal nucleic acid amplification tests (NAATs) paired with electroanalytical detection enable cost-effective, sensitive, and specific digital pathogen detection for various in situ applications such as point-of-care medical diagnostics, food safety monitoring, and environmental surveillance. Self-assembled monolayers (SAMs) on gold surfaces are reliable platforms for electroanalytical DNA biosensors. However, the lack of automation and scalability often limits traditional chip-based systems. To address these challenges, we propose a continuous thread-based device that enables multiple electrochemical readings on a functionalized working electrode Au thread with a single connection point. We demonstrate the possibility of rolling the thread on a spool, which enables easy manipulation in a roll-to-roll architecture for high-throughput applications. As a proof of concept, we have demonstrated the detection of recombinase polymerase amplification (RPA) isothermally amplified DNA from the two toxic microalgae species Ostreopsis cf. ovata and Ostreopsis cf. siamensis by performing a sandwich hybridization assay (SHA) with electrochemical readout. [ABSTRACT FROM AUTHOR]

Nucleic acids are prominent biomarkers for diagnosing infectious pathogens using nucleic acid amplification techniques (NAATs). PCR, a gold standard technique for amplifying nucleic acids, is widely used in scientific research and diagnosis. Efficient pathogen detection is a key to adequate food safety and hygiene. However, using bulky thermal cyclers and costly laboratory setup limits its uses in developing countries, including India. The isothermal amplification methods are exploited to develop miniaturized sensors against viruses, bacteria, fungi and other pathogenic organisms and have been applied for in situ diagnosis. Isothermal amplification techniques have been found suitable for POC techniques and follow WHO's ASSURED criteria. LAMP, NASBA, SDA, RCA and RPA are some of the isothermal amplification techniques which are preferable for POC diagnostics. Furthermore, methods such as WGA, CPA, HDA, EXPAR, SMART, SPIA and DAMP were introduced for even more accuracy and robustness. Using recombinant polymerases and other nucleic acid-modifying enzymes has dramatically broadened the detection range of target pathogens under the scanner. The coupling of isothermal amplification methods with advanced technologies such as CRISPR/Cas systems, fluorescence-based chemistries, microfluidics and paper-based sensors has significantly influenced the biosensing and diagnosis field. This review comprehensively analyzed isothermal nucleic acid amplification methods, emphasizing their advantages, disadvantages and limitations. [ABSTRACT FROM AUTHOR]

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), and strand-displacing DNA polymerase (Pol). Component instability and the need to store commercial kits in a deep freezer until use are some limitations of RPA. In a previous study, Bacillus stearothermophilus Pol (Bst -Pol) was used as a thermostable strand-displacing DNA polymerase in RPA. Here, we attempted to optimize the lyophilization conditions for RPA with newly isolated thermostable DNA polymerases for storage at room temperature. We isolated novel two thermostable strand-displacing DNA polymerases, one from a thermophilic bacterium Aeribacillus pallidus (H1) and the other from Geobacillus zalihae (C1), and evaluated their performances in RPA reaction. Urease subunit β (UreB) DNA from Ureaplasma parvum serovar 3 was used as a model target for evaluation. The RPA reaction with H1-Pol or C1-Pol was performed at 41 °C with the in vitro synthesized standard UreB DNA. The minimal initial copy numbers of standard DNA from which the amplified products were observed were 600, 600, and 6000 copies for RPA with H1-Pol, C1-Pol, and Bst -Pol, respectively. Optimization was carried out using RPA components, showing that the lyophilized RPA reagents containing H1-Pol exhibited the same performance as the corresponding liquid RPA reagents. In addition, lyophilized RPA reagents with H1-Pol showed almost the same activity after two weeks of storage at room temperature as the freshly prepared liquid RPA reagents. These results suggest that lyophilized RPA reagents with H1-Pol are preferable to liquid RPA reagents for onsite use. [ABSTRACT FROM AUTHOR]

Analytical systems based on isothermal nucleic acid amplification tests (NAATs) paired with electroanalytical detection enable cost-effective, sensitive, and specific digital pathogen detection for various in situ applications such as point-of-care medical diagnostics, food safety monitoring, and environmental surveillance. Self-assembled monolayers (SAMs) on gold surfaces are reliable platforms for electroanalytical DNA biosensors. However, the lack of automation and scalability often limits traditional chip-based systems. To address these challenges, we propose a continuous thread-based device that enables multiple electrochemical readings on a functionalized working electrode Au thread with a single connection point. We demonstrate the possibility of rolling the thread on a spool, which enables easy manipulation in a roll-to-roll architecture for high-throughput applications. As a proof of concept, we have demonstrated the detection of recombinase polymerase amplification (RPA) isothermally amplified DNA from the two toxic microalgae species Ostreopsis cf. ovata and Ostreopsis cf. siamensis by performing a sandwich hybridization assay (SHA) with electrochemical readout.

Diagnostic tests for brown spider accidents are unavailable and impact treatment decisions, increasing costs and patient risks. In this work, we used for the first time a fast, simple, and visual method based on the loop-mediated isothermal amplification assay (LAMP) to detect Loxosceles envenomation. Using the DNA from L. similis legs, we observed a high sensitivity using this test since as low as 0.32 pg of DNA could be detected. This pH-dependent colorimetric assay was 64 times more sensitive than PCR to detect spider DNA. The test was specific for Loxosceles once no cross-reaction was observed when testing DNA from different agents that cause similar dermonecrotic injuries. The test allowed the detection of Loxosceles intermedia DNA from hair, serum, and exudate samples obtained from experimentally-envenomed rabbit within 72 h. The method sensitivity varied according to the sample and the collection time, reaching 100% sensitivity in serum and hair, respectively, 1 h and 24 h after the experimental envenomation. Due to its ease of execution, speed, sensitivity, and specificity, LAMP presents an excellent potential for identifying Loxosceles spp. Envenomation. This can reduce the burden on the Health System and the morbidity for the patient by implementing the appropriate therapy immediately.In addition, this work opens up the perspective to other venomous animal accident identification using LAMP. [Display omitted] • Using 28S primers it was possible to identify L. similis' DNA with high sensitivity. • LAMP was 62-fold more sensitive than PCR and detected as low as 0.32 pg of DNA. • LAMP detected L. intermedia DNA from hair, serum, and exudate from experimentally-envenomed rabbits. • LAMP presents an excellent potential for identifying Loxosceles spp. Envenomation. [ABSTRACT FROM AUTHOR]

Detection of CpG methylation levels holds immense potential for application in medical diagnosis of various diseases. In this study, we report the development of a recombinase polymerase amplification (RPA)-based CpG methylation level sensing system on G-quadruplex (G4) and intercalated motif (i-motif)-forming regions, which are stabilized by CpG methylation. This detection system is based on the principle that DNA polymerase is stalled at the methylated G4 and i-motif-forming region, which results in a decrease in the initial elongation efficiency of RPA. This reduction in turn affects the onset of amplification depending on the extent of CpG methylation; therefore, the methylation level is quantified by RPA. We demonstrate that the onset of amplification was delayed by CpG methylation when PCR products containing the vascular endothelial growth factor (VEGF) G4 and i-motif-forming region were used as the template. Furthermore, onset of amplification was delayed with the increase in CpG methylation of the VEGF region on genomic DNA. These results demonstrate that the sensing system is capable of directly detecting the methylation level at a constant temperature (39 °C) within 30 min without performing bisulfite conversion or affinity capture of methylated DNA. [ABSTRACT FROM AUTHOR]

The wheat crop is one of the most cultivated and consumed commodities all over the world. Fungal diseases are of particular concern for wheat cultivation since they cause great losses and reduced quality, and also for the accumulation of toxin compounds into the final product. In this scenario, optimal disease management strategies are a key point to boosting food production and sustainability in agriculture. Innovative and point-of-care diagnostic technologies represent a powerful weapon for early detection of fungal pathogens and preventively counteract diseases on wheat with the aim to drastically reduce the fungicides as inputs. Indeed, in-field diagnostics devices are fast, sensitive, and ready-to-use technologies able to promptly detect a low inoculum concentration even at the pre-symptomatic stage of the disease. Promising isothermal molecular and phenomics-based methods have been developed to detect wheat fungal pathogens directly in the field. Such technologies could be potentially coupled to directly detect the presence of a certain pathogen and indirectly disclose the plant-pathogen interactions since spectral-based methodologies detect host perturbations following the infection. The present review reports the main in-field isothermal molecular-based and phenomics-based detection technologies for fungal pathogens in wheat discussing their advantages, disadvantages, and potential applications in the near future.

Hepatozoonosis, caused by the protozoan Hepatozoon canis , is a prevalent blood disease affecting owned and stray dogs and cats. The prevalence of these parasites among companion animals in Thailand remains poorly understood. Diagnosing the old-world form of the disease is challenging due to the wide range of nonspecific clinical signs and the reliance on finding low levels of Hepatozoon gamonts in blood smears for conventional diagnosis. PCR demonstrates high specificity and sensitivity but it requires sophisticated instrumentation. Therefore, we established recombinase polymerase amplification (RPA) coupled with Cas12a for H. canis detection based on 18S rRNA. Our findings showed that RPA-Cas12a using gRNA_H was highly specific to H. canis , without yielding positives for other pathogen species including Babesia species. Even in cases of co-infection, RPA-Cas12a only detected positives in samples containing H. canis. This approach detected minimal amounts of H. canis 18S rRNA-harboring plasmid at 10 copies per reaction, whereas plasmid-spiked canine blood enabled detection at a minimal amount of 100 copies per reaction. The performance of RPA-Cas12a was validated by comparing it with quantitative PCR-high resolution melting analysis (qPCR-HRM) and sequencing based on 35 canine blood samples. RPA-Cas12a demonstrated precision and accuracy values of 94 % and 90 %, respectively comparable to qPCR-HRM. Overall, these results indicate that RPA-Cas12a serves as a promising tool for H. canis detection as indicated by comparable performance to qPCR-HRM and is suitable for implementation in small animal hospitals or clinics due to its minimal resource requirements, thereby contributing to effective diagnosis and treatment for infected dogs. [Display omitted] • RPA-Cas12a offers a rapid and accurate approach for H. canis detection. • RPA-Cas12a can specifically detect H. canis without cross reaction. • LOD was at 10 and 100 copies in direct plasmid and spiked canine blood, respectively. • RPA-Cas12a achieve to apply with DNA obtained from clinical blood. • RPA-Cas12a display precision and accuracy comparable to qPCR-HRM. [ABSTRACT FROM AUTHOR]

Sickle cell disease (SCD) is the most prevalent life-threatening blood monogenic disorder. Currently, there is no cure available, apart from bone marrow transplantation. Early and efficient diagnosis of SCD is key to disease management, which would make considerable strides in alleviating morbidity and reducing mortality. However, the cost and complexity of diagnostic procedures, such as the Sanger sequencing method, impede the early detection of SCD in a resource-limited setting. To address this, the current study demonstrates a simple and efficient proof-of-concept assay for the detection of patients and carriers using extraction-free non-invasive buccal swab samples by isothermal DNA Amplification coupled Restrictase-mediated cleavage (iDAR). This study is a first of its kind reporting the use of buccal swab specimens for iDA in molecular diagnosis of a genetic disease, all the while being cost effective and time saving, with the total assay time of around 150 min at a cost of USD 5. Further, iDAR demonstrates 91.5% sensitivity and 100% specificity for detecting all three alleles: SS, AS, and AA, having a 100% concordance with Sanger sequencing. The applicability of the iDAR assay is further demonstrated with its adaptation to a one-pot reaction format, which simplifies the assay system. Overall, iDAR is a simple, cost-effective, precise, and non-invasive assay for SCD screening, with the potential for use in a limited resource setting. [ABSTRACT FROM AUTHOR]

Background: Recombinase (uvsY and uvsX) from bacteriophage T4 is a key enzyme for recombinase polymerase amplification (RPA) that amplifies a target DNA sequence at a constant temperature with a single-stranded DNA-binding protein and a strand-displacing polymerase. The present study was conducted to examine the effects of the N- and C-terminal tags of uvsY on its function in RPA to detect SARS-CoV-2 DNA. Methods: Untagged uvsY (uvsY-Δhis), N-terminal tagged uvsY (uvsY-Nhis), C-terminal tagged uvsY (uvsY-Chis), and N- and C-terminal tagged uvsY (uvsY-NChis) were expressed in Escherichia coli and purified. RPA reaction was carried out with the in vitro synthesized standard DNA at 41 °C. The amplified products were separated on agarose gels. Results: The minimal initial copy numbers of standard DNA from which the amplified products were observed were 6 × 105, 60, 600, and 600 copies for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The minimal reaction time at which the amplified products were observed were 20, 20, 30, and 20 min for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The RPA with uvsY-Nhis exhibited clearer bands than that with either of other three uvsYs. Conclusions: The reaction efficiency of RPA with uvsY-Nhis was the highest, suggesting that uvsY-Nhis is suitable for use in RPA. [ABSTRACT FROM AUTHOR]

Purpose: Colchicum autumnale (Colchicaceae) exhibits toxicity, and severe poisoning cases due to ingestion of this plant have been reported in Japan. Identifying the cause of poisoning is important for emergency medical treatment, and a rapid and simple detection technique is required for the identification of the cause of poisoning. In the present study, we developed a rapid and simple method for the detection of C. autumnale based on a loop-mediated isothermal amplification (LAMP) assay, which is 100- to 1000-fold higher than the conventional polymerase chain reaction method. Methods: Specific LAMP primers for C. autumnale were designed based on the trnL-trnF intergenic spacer region. Using the LAMP primers, the LAMP assay was performed at an isothermal reaction temperature of 63 ℃. Results: The LAMP reaction was shown to be specific and highly sensitive to C. autumnale, given that the assay can be used for 10 pg of purified DNA. Using a simple protocol for on-site detection, the entire procedure from pretreatment to evaluation required approximately 1 h. Moreover, the LAMP method using a microfluidic device detected multiple genes, including C. autumnale-specific DNA regions, at a time. Conclusions: The currently proposed protocol exhibits good potential as a screening method for C. autumnale poisoning in emergency medical care. [ABSTRACT FROM AUTHOR]

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. Image 1 • Single enzyme-based stem-loop and linear primers co-mediated exponential amplification (SLIMP) is a novel isothermal DNA amplification only recognizing two target sites. • SLIMP can efficiently amplify short gene sequence with the concentration down to 1 aM. • SLIMP can quantify the short gene sequences with concentrations ranging from 10 nM to 10 fM. • SLIMP has high specificity and good selectivity as well as the feasibility for real time test. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) in a blood sample are usually measured by quantitative reverse transcription PCR (qRT-PCR), microarray, and next-generation sequencing (NGS) which requires time-consuming pre-treatment, manual operation, and a stand-alone instrument. To overcome these disadvantages, miRNA testing has been developed using the automated analyzers routinely used in clinical laboratories. An isothermal DNA amplification reaction was adapted to a fully automated immunoassay analyzer that conducts extraction, amplification, and detection processes at 37 °C in 44 min. In a reaction vessel, a pre-designed single-stranded signal DNA was amplified in the presence of miRNA, using DNA templates, DNA polymerase, and nicking endonuclease. Then, the amplified signal DNA was hybridized by one DNA probe attached to a magnetic particle and another DNA probe labeled with acridinium ester. After the chemiluminescence reaction, luminescence intensity was automatically measured. The automated assays of cancer-related miRNAs were implemented on the analyzer with throughput of 66 tests per hour. In the assays with one-step amplification, three miRNAs (miR-21-5p, miR-18a-5p, and miR-500a-3p) at concentrations lower than 100 fM were automatically detected and the cross reactivity for miR-21-5p with fifteen similar miRNAs was not higher than 0.02%. In the assay with two-step amplification, detection sensitivity and amplification rate for miR-21-5p were 3 fM and 103-fold, respectively. The coefficient of variations (CVs) in the measurement at the target concentrations from 5 fM to 1000 pM were less than 8%. Furthermore, we also achieved automated nucleic acid detection in human serum. The proposed fully automated miRNA assays showed high sensitivity, low cross reactivity, and reproducibility suitable for clinical use. [ABSTRACT FROM AUTHOR]

Abstract Consumer electronic technologies offer practical performances to develop compact biosensing systems intended for the point-of-care testing of DNA biomarkers. Herein a discrimination method for detecting single nucleotide polymorphisms, based on isothermal amplification and on-chip hybridisation, was developed and integrated into user-friendly optical devices: e.g., USB digital microscope, flatbed scanner, smartphone and DVD drive. In order to adequately identify a single base change, loop-mediated isothermal amplification (LAMP) was employed, with high yields (8 orders) within 45 min. Subsequently, products were directly hybridised to the allele-specific probes attached to plastic chips in an array format. After colorimetric staining, four consumer electronic techniques were compared. Sensitive precise measurements were taken (high signal-to-noise ratios, 10-μm image resolution, 99% scan-to-scan reproducibility). These features confirmed their potential as analytical tools, are a competitive alternative to fluorescence scanners, and incorporate additional advantages, such as user-friendly interface and connectivity for telemedicine needs. The analytical performances of the integrated platform (assay and reader) in the human samples were also excellent, with a low detection limit (100 genomic DNA copies), and reproducible (<15%) and cheap assays (< 10 €/test). The correct genotyping of a genetic biomarker (single-nucleotide polymorphism located in the GRIK4 gene) was achieved as the assigned genotypes agreed with those determined by using sequencing. The portability, favourable discriminating and read-out capabilities reveal that the implementation of mass-produced low-cost devices into minimal-specialised clinical laboratories is closer to becoming a reality. Graphical abstract fx1 Highlights • Demonstration of consumer electronic systems as alternative bioanalytical readers. • Performance comparison of camera-based and scanning devices. • Point-of-care testing in DNA array format. • Accurate genotyping for pharmacogenomics predictions. [ABSTRACT FROM AUTHOR]

Highlights • Glanders, primarily a disease of equines needs early diagnosis for timely disease management in humans and infection containment in animals. • The present LAMP assay rapidly detects B. mallei obviating the requirement of thermal cycler. • The assay can detect as low as 250 fg genomic DNA of B. mallei. • The assay presents the detection of B. mallei in artificially spiked blood and tap water samples. • LAMP assay can be a viable alternative to the PCR based detection of B. mallei in glanders endemic regions with resource-limited settings. Abstract Burkholderia mallei, a potential biological warfare agent, is the causative agent of an infectious, fatal, and zoonotic disease, called glanders. Accurate and early diagnosis of glanders is important to control the disease lethality and infection spread. Molecular detection of B. mallei is considered strenuous because B. mallei is a subtractive genomic clone of B. pseudomallei. The present study was aimed at development of a real-time LAMP assay for detection of B. mallei. The LAMP assay was highly sensitive and could detect ≥250 fg of genomic DNA of B. mallei and ≥100 copies of recombinant plasmid containing target DNA sequence. In artificially spiked blood and water samples, it could detect ≥2.1 × 103 and ≥4.73 × 102 CFU/mL of B. mallei, respectively. The assay was highly specific for B. mallei as none of the other bacteria used in the study tested positive. The reported LAMP assay being simple and rapid can be a viable alternative to PCR-based glanders diagnostic assays in glanders endemic regions with resource-limited settings. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Fabrication, and evaluation of an integrated Lab-on-Chip platform. • Bacteria capture, thermal lysis, DNA purification, and isothermal DNA amplification on chip. • Pathogen screening in milk samples for cell lysates corresponding to <500 cells. Abstract Strict regulations govern food safety as the latter is of extremely high importance and a major public issue, while it is demanding in respect of analytical effort. The duration of common food analysis practices is typically 2–5 days. Microfluidics can reduce labor and analysis time to a few hours, proving its beneficial use and rendering the response of food industry as well as public safety agents faster. The aim of the present work is the fabrication and evaluation of a modular integrated Lab-on-Chip platform for sample preparation, accommodating bacteria capture, thermal lysis, DNA purification, and isothermal DNA amplification, addressing pathogen screening in milk samples for cell lysates corresponding to <500 cells. We note that individual modules have been tested to work down to 10 cells. [ABSTRACT FROM AUTHOR]

Molecular diagnostic tools in the field of food and water quality analysis are becoming increasingly widespread. Usually, based on DNA amplification techniques such as polymerase chain reaction (PCR), these methods are highly sensitive and versatile but require well-equipped laboratories and trained personnel. To reduce analysis time and avoid expensive equipment, isothermal DNA amplification methods for detecting various target organisms have been developed. However, to make molecular diagnostics suitable for low-resource settings and in-field applications, it is crucial to continuously adapt the working steps associated with DNA amplification, namely sample preparation, DNA extraction, and visualization of the results. Many novel approaches have been evaluated in recent years to tackle these challenges, e.g., the use of ionic liquids for the rapid isolation of nucleic acids from organisms relevant for food and water analysis or the integration of entire analytical workflows on microfluidic chips. In any event, the future of applications in the field of isothermal amplification will probably lie in ready-to-use cartridges combined with affordable handheld devices for on-site analysis. This trend article aims to make prospective users more familiar with this technology and its potential for moving molecular diagnostics from the laboratory to the field. Graphical abstract ᅟ [ABSTRACT FROM AUTHOR]

The easy and rapid spread of bacterial contamination and the risk it poses to human health makes evident the need for analytical methods alternative to conventional time-consuming laboratory-based techniques for bacterial detection. To tackle this demand, biosensors based on isothermal DNA amplification methods have emerged, which avoid the need for thermal cycling, thus facilitating their integration into small and low-cost devices for in situ monitoring. This review focuses on the breakthroughs made on biosensors based on isothermal amplification methods for the detection of bacteria in the field of food safety and environmental monitoring. Optical and electrochemical biosensors based on loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), strand displacement amplification (SDA), and isothermal strand displacement polymerisation (ISDPR) are described, and an overview of their current advantages and limitations is provided. Although further efforts are required to harness the potential of these emerging analytical techniques, the coalescence of the different isothermal amplification techniques with the wide variety of biosensing detection strategies provides multiple possibilities for the efficient detection of bacteria far beyond the laboratory bench.

Abstract The recently developed methods of nucleic acids isothermal amplification are promising tools for point-of-care diagnostics and in the field detection of pathogenic microorganisms. However, application of these methods outside a laboratory faces some challenges such as the rapid and sensitive detection of amplified products and the absence of cross-reactivity with genetically related microorganisms. In the presented study we compared three methods of isothermal DNA amplification loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) and thermophilic helicase-dependent isothermal DNA amplification (tHDA), for detection of highly dangerous pathogens, such as Bacillus anthracis, Francisella tularensis and Yersinia pestis , and combined them with lateral flow dipsticks for the rapid visualization of amplified products. We observed low specificity of the three methods for B. antharcis , medium for Y. pestis and high for F. tularensis detection. Sensitivity and the detection limit were high and comparable for all the methods. We concluded that the lateral flow dipsticks have been a very useful tool for product detection of the isothermal amplification methods and enable reading the results without the use of any equipment. However, our results showed that the use of isothermal amplification methods is strongly related to the risk of false positive results. Highlights • The use of lateral flow dipsticks for rapid, sensitive and equipment-free visualization of LAMP, tHDA and RPA results. • Limited specificity of isothermal nucleic acids amplification methods for some pathogens. • High sensitivity and the detection limit of LAMP, tHDA and RPA methods applied for B. antharcis , Y. pestis and F. tularensis. [ABSTRACT FROM AUTHOR]

Recent zoonotic outbreaks, such as Zika, Middle East respiratory syndrome and Ebola, have highlighted the need for rapid and accurate diagnostic assays that can be used to aid pathogen control. Q fever is a zoonotic disease caused by the transmission of Coxiella burnetii that can cause serious illness in humans through aerosols and is considered a potential bioterrorism agent. However, the existing assays are not suitable for the detection of this pathogen due to its low levels in real samples. We here describe a rapid bio‐optical sensor for the accurate detection of Q fever and validate its clinical utility. By combining a bio‐optical sensor, that transduces the presence of the target DNA based on binding‐induced changes in the refractive index on the waveguide surface in a label‐free and real‐time manner, with isothermal DNA amplification, this new diagnostic tool offers a rapid (<20 min), 1‐step DNA amplification/detection method. We confirmed the clinical sensitivity (>90%) of the bio‐optical sensor by detecting C. burnetii in 11 formalin‐fixed, paraffin‐embedded liver biopsy samples from acute Q fever hepatitis patients and in 16 blood plasma samples from patients in which Q fever is the cause of fever of unknown origin. [ABSTRACT FROM AUTHOR]

Detecting, monitoring, and mapping transgenic sequences in the environment has been a long-standing challenge in ecology. Given the level of social, economic, and scientific interest in them, transgenic (GM) DNA sequences stand as paradigmatic; such sequences further have the unique property of functioning as unequivocal markers in the environment, clearly showing the paths of gene flow between crops and their wild relatives, and across industrial and traditional forms of agriculture. True mapping of transgenic DNA at the landscape level requires a method of DNA amplification that is robust and isothermal in order to be cost effective and field-based. We present here a method with these characteristics. Our multiplex fluorescent method is for the detection of the p35s CaMV promoter frequently used in transgenic plants as well as the alpha-zein protein specific to maize, but can be applicable to any other DNA sequence. The method uses loop-mediated isothermal amplification (LAMP) with visualization achieved using a fluorophore-quencher system of DNA hybridizing probes. We demonstrate the applicability of this tri-color method to transgenic corn, non-transgenic corn, and non-transgenic/non-corn species. [ABSTRACT FROM AUTHOR]

Summary: Burkholderia mallei is the aetiological agent of glanders, a highly contagious and re‐emerging zoonotic disease. Early diagnosis of glanders is critically important to ensure timely treatment with appropriate antibiotics in humans, and to prevent spread of infection in animals. Molecular detection of B. mallei has always been troublesome because of its genetic similarity with Burkholderia pseudomallei, the causative agent of melioidosis. In present investigation, a set of six B. mallei‐specific primers were designed and a simple, rapid, specific and sensitive real‐time loop‐mediated isothermal amplification (LAMP) assay was developed for detection of B. mallei. The LAMP assay could detect as low as 1 pg of B. mallei genomic DNA and 5.5 × 103 CFU/ml of B. mallei in spiked human blood. The assay was highly specific for B. mallei as it did not cross‐react with other bacterial strains used in the study. The established LAMP assay is field adaptable and can be a better and viable alternative to PCR‐based techniques for detection of B. mallei in glanders endemic areas with resource‐limited settings. [ABSTRACT FROM AUTHOR]

Plasmodiophora brassicae, the cause of clubroot, is a very serious problem preventing from successful and profitable cultivation of oilseed rape in Poland. The pathogen was found in all main growing areas of oilseed rape; it also causes considerable problems in growing of vegetable brassicas. The aim of this work was to elaborate fast, cheap and reliable screening method to detect P. brassicae. To achieve this aim the Loop-mediated isothermal DNA amplification (LAMP) technique has been elaborated. The set of three primer pairs was designed using LAMP software. The detection was performed with the GspSSD polymerase, isolated from bacteria Geobacillus sp., with strand displacement activity. DNA extraction from clubbed roots obtained from farmers’ fields of oilseed rape infected by P. brassicae was done using a modified CTAB method. The reaction was performed for 60 min at 62oC. The visual detection was done using CFX96 Real Time PCR Detection System (BioRad) or Gerie II Amplicatior (Optigen). The detection with LAMP proved its usefulness; it was easy, fast and accurate and independent of plant age. The detection limit was 5 spores per 1 µl of the spore suspension, so LAMP was less sensitive than quantitative PCR tests reported in the literature. However, the method is cheap and simple, so it is a good alternative, when it comes to practical use and the assessment of numerous samples.

Loop-mediated isothermal amplification (LAMP) is a rapid and reliable sequence-specific isothermal nucleic acid amplification technique. To date, all reported real-time detection methods for LAMP have been restricted to single targets, limiting the utility of this technique. Here, we adapted standard LAMP primers to contain a quencher-fluorophore duplex region that upon strand separation results in a gain of fluorescent signal. This approach permitted the real-time detection of 1–4 target sequences in a single LAMP reaction tube utilizing a standard real-time fluorimeter. The methodology was highly reproducible and sensitive, detecting below 100 copies of human genomic DNA. It was also robust, with a 7-order of magnitude dynamic range of detectable targets. Furthermore, using a new strand-displacing DNA polymerase or its warm-start version, Bst 2.0 or Bst 2.0 WarmStart DNA polymerases, resulted in 50% faster amplification signals than wild-type Bst DNA polymerase, large fragment in this new multiplex LAMP procedure. The coupling of this new multiplex technique with next generation isothermal DNA polymerases should increase the utility of the LAMP method for molecular diagnostics.

In DNA amplification, the initial step of copying a target sequence from the template DNA, or the so-called intermediate product generation step, is very important. In examining the turn-back primer (TP)–dependent isothermal DNA amplification (TIA) method, we determined the actual time point of intermediate product generation by extrapolating dsDNA amplification curves. Our results indicate that intermediate product creation is the rate-limiting step in TIA, and good TP design is advantageous for improving the intermediate production process.

Isothermal methods, such as helicase-dependent amplification (HDA), have an advantage over polymerase chain reaction for DNA amplification owing to their ease of operation. Here, we developed a new HDA method that is nanoparticle-assisted, termed nanoHDA. This method uses gold nanoparticles (AuNPs) to improve the sensitivity and specificity of the isothermal method. In HDA, the denaturation of DNA templates is mediated by helicases, but this method is limited by the low denaturation efficiency of helicases. In this report, AuNPs with preferential affinity for single-stranded DNA (ssDNA) were utilized to improve the denaturation efficiency of helicases. The same affinity property of nanoparticles can also enhance specificity by suppressing primer-dimer formation. This nanoHDA method was employed to genotype the KRAS gene in genomic DNA samples from colorectal cancer patients, as achieved by the hybridization of nanoHDA amplicons using the NanoBioArray chip. [ABSTRACT FROM AUTHOR]

Rolling circle amplification (RCA) developed in the mid-1990s has been widely used as an efficient isothermal DNA amplification process for molecular diagnosis. This enzymatic process amplifies target DNA sequences with high fidelity and specificity by using the strand displacing DNA polymerases. The product of RCA is long single-stranded DNA that contains tandem repeat of target sequence. Isothermal reaction amplification condition of RCA has an advantage over conventional polymerase chain reaction, because no temperature cycling devices are needed for RCA. Thus, RCA is suitable tool for point-of-care detection of target nucleic acids as well as facile detection of target genes. Combined with various detection methods, RCA could amplify and detect femtomolar scale of target nucleic acids with a specificity of one or two base discrimination. Herein, RCA technology is reviewed with an emphasis on molecular diagnosis of microRNAs, infectious pathogens, and point mutations. [ABSTRACT FROM AUTHOR]

Rapid, early, and accurate diagnosis of malaria is essential for effective disease management and surveillance, and can reduce morbidity and mortality associated with the disease. Although significant advances have been achieved for the diagnosis of malaria, these technologies are still far from ideal, being time consuming, complex and poorly sensitive as well as requiring separate assays for sample processing and detection. Therefore, the development of a fast and sensitive method that can integrate sample processing with detection of malarial infection is desirable. Here, we report a two-stage sample-to-answer system based on nucleic acid amplification approach for detection of malaria parasites. It combines the Dimethyl adipimidate (DMA)/Thin film Sample processing (DTS) technique as a first stage and the Mach-Zehnder Interferometer-Isothermal solid-phase DNA Amplification (MZI-IDA) sensing technique as a second stage. The system can extract DNA from malarial parasites using DTS technique in a closed system, not only reducing sample loss and contamination, but also facilitating the multiplexed malarial DNA detection using the fast and accurate MZI-IDA technique. Here, we demonstrated that this system can deliver results within 60 min (including sample processing, amplification and detection) with high sensitivity (<1 parasite μL −1 ) in a label-free and real-time manner. The developed system would be of great potential for better diagnosis of malaria in low-resource settings. [ABSTRACT FROM AUTHOR]

Epidermal growth factor receptor ( EGFR ) is a non-small-cell lung cancer biomarker, based on which several near-patient-testing methods have been developed and applied to predict treatment response on individual patients. Existing methods for detection of EGFR mutation are costly, labor-intensive and time-consuming. In this paper, we report a novel EGFR mutation testing system, which is based on Mach–Zehnder Interferometer (MZI) sensor and isothermal solid-phase DNA amplification (IDA) technique, called MZI-IDA sensor system. The system can deliver results within 30 min and shows high sensitivity to detect trace amounts of genomic DNA (<1 copy). In addition, the system is able to detect a L858R mutation in a 99:1 mixture of wild-type and mutant cells. In a pilot clinical study, the system is compared with conventional methods (PCR and direct sequencing) by using tissue biopsy samples from NSCLC patients. The MZI-IDA sensor system is proved to be capable of fast and accurate detection of the L858R mutation of EGFR gene in clinical samples. This may greatly help the clinicians develop an appropriate treatment plan. [ABSTRACT FROM AUTHOR]

DNA polymerases (DNAPs) responsible for genome replication are highly faithful enzymes that nonetheless cannot deal with damaged DNA. In contrast, translesion synthesis (TLS) DNAPs are suitable for replicating modified template bases, although resulting in very low-fidelity products. Here we report the biochemical characterization of the temperate bacteriophage Bam35 DNA polymerase (B35DNAP), which belongs to the protein-primed subgroup of family B DNAPs, along with phage Φ29 and other viral and mobile element polymerases. B35DNAP is a highly faithful DNAP that can couple strand displacement to processive DNA synthesis. These properties allow it to perform multiple displacement amplification of plasmid DNA with a very low error rate. Despite its fidelity and proofreading activity, B35DNAP was able to successfully perform abasic site TLS without template realignment and inserting preferably an A opposite the abasic site (A rule). Moreover, deletion of the TPR2 subdomain, required for processivity, impaired primer extension beyond the abasic site. Taken together, these findings suggest that B35DNAP may perform faithful and processive genome replication in vivo and, when required, TLS of abasic sites. [ABSTRACT FROM AUTHOR]

A valid solution for micro-analytical systems is the selection of a compatible amplification reaction with a simple, highly-integrated efficient design that allows the detection of multiple genomic targets. Two approaches under isothermal conditions are presented: recombinase polymerase amplification (RPA) and multiple displacement amplification (MDA). Both methods were applied to a duplex assay specific for Salmonella spp . and Cronobacter spp., with excellent amplification yields (0.2–8.6 · 10 8 fold). The proposed approaches were successfully compared to conventional PCR and tested for the milk sample analysis as a microarray format on a compact disc (support and driver). Satisfactory results were obtained in terms of resistance to inhibition, selectivity, sensitivity (10 1 –10 2 CFU/mL) and reproducibility (below 12.5%). The methods studied are efficient and cost-effective, with a high potential to automate microorganisms detection by integrated analytical systems working at a constant low temperature. [ABSTRACT FROM AUTHOR]

LAMP is an innovative, simple, rapid, specific and cost-effective nucleic acid amplification method. Due to the use of a special enzyme – GspSSD polymerase, the reaction takes a short time and can be performed at isothermal conditions. The sensitivity and specificity of LAMP technique is significantly higher, than standard PCR techniques, as two or three specific primer pairs are used. The technique is regarded as a useful tool for the detection and identification of plant pathogens. In this work, LAMP was used to study the composition of the population of fungi of the genus Leptosphaeria, causing a damaging disease of oilseed rape, called blackleg or stem canker. The detection concerned DNA present in fungal spores contained in air samples obtained using Hirst-type volumetric trap, in Pomerania (north Poland) in 2010. The results achieved using the LAMP technique were similar to these obtained with previously used, highly specific method of Real-time PCR. Conducting LAMP reaction was much easier and less time-and cost-consuming, due to a simplified method of DNA isolation of pathogens from plant tissues. Then, the LAMP technique was used to assess the composition of the population of Leptosphaeria spp. in plants of oil- seed rape collected from the field in the Opole region (south-we- stern part of Poland) in 2013. In contrast to studies conducted in 2002–2003, the analysis of leaf symptoms showed a higher pro- portion of L. maculans compared to L. biglobosa, what reflects changes in the composition of pathogen population of fungi causing blackleg on oilseed rape in this part of Poland.

Copyright of Acta Agrobotanica is the property of Polish Botanical Society / Polskie Towarzystwo Botaniczne and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract: Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for single-base mutations in biological samples. [Copyright &y& Elsevier]

Copyright of Acta Agrobotanica is the property of Polish Botanical Society / Polskie Towarzystwo Botaniczne and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

You spin me round: Using a destabilizing abasic site and high concentration of ligase, rapid DNA self‐replication in an isothermal ligase chain reaction (LCR) was produced. Both destabilization and rapid ligation are essential for proper LCR replication. This method also provides insight into prebiotic nucleotide replication and is a potential amplification method for biodiagnostics. [ABSTRACT FROM AUTHOR]

Abstract: Here, we introduce an integrated biochip which offers accurate thermal control and sensitive electrochemical detection of DNA amplification in real-time. The biochip includes a 10-μl microchamber, a temperature sensor, a heater, and a contactless impedance biosensor. A pair of interdigitated electrodes is employed as the impedance biosensor and the products of the amplification are determined directly through tracing the impedance change, without using any labels, redox indicators, or probes. Real-time monitoring of strand-displacement amplification and rolling circle amplification was successfully performed on the biochip and a detection limit of 1nM was achieved. Amplification starting at an initial concentration of 10nM could be discriminated from that starting at 1nM started concentration as well as from the negative control. Since an insulation layer covers the electrodes, the electrodes are spared from erosion, hydrolysis and bubble formation on the surface, thus, ensuring a long lifetime and a high reusability of the sensor. In comparison to bench-top apparatus, our chip shows good efficiency, sensitivity, accuracy, and versatility. Our system requires only simple equipments and simple skills, and can easily be miniaturized into a micro-scale system. The system will then be suitable for a handheld portable device, which can be applied in remote areas. It covers merits such as low cost, low-power consumption, rapid response, real-time monitoring, label-free detection, and high-throughput detection. [Copyright &y& Elsevier]

A new electrochemical sequence-specific DNA detection platform based on primer generation-rolling circle amplification (PG-RCA), methylene blue (MB) redox indicator, and indium tin oxide (ITO) electrode is reported. In the presence of a specific target sequence, PG-RCA, an isothermal DNA amplification technique, produced large amounts of amplicons in an exponential manner. In addition to the standard components, the reaction mixture contained MB, which bound with the PG-RCA amplicons. End-point electrochemical measurement by differential pulse voltammetry (DPV) was performed using ITO electrode. The amplicon-bound MB resulted in a lower DPV signal than free MB due to a smaller diffusion coefficient as well as electrostatic repulsion between the negatively charged amplicon-bound MB and ITO electrode. With simple assay design (recognition probe) and instrumentation (operating temperature at 37 °C and ITO electrode without the need for probe immobilization), this detection platform is well suited for point-of-care and on-site testing. Real-time measurement was also achieved by pretreating the ITO electrode with bovine serum albumin. [ABSTRACT FROM AUTHOR]

Abstract: The present study describes the development of a DNA based biosensor to detect Mycobacterium tuberculosis using thermophilic helicase-dependent isothermal amplification (tHDA) and dextrin coated gold nanoparticles (AuNPs) as electrochemical reporter. The biosensor is composed of gold nanoparticles (AuNPs) and amine-terminated magnetic particles (MPs) each functionalized with a different DNA probe that specifically hybridize with opposite ends of a fragment within the IS6110 gene, which is M. tuberculosis complex (MTC) specific. After hybridization, the formed complex (MP-target-AuNP) is magnetically separated from the solution and the AuNPs are electrochemically detected on a screen printed carbon electrode (SPCE) chip. The obtained detection limit is 0.01ng/μl of isothermally amplified target (105bp). This biosensor system can be potentially implemented in peripheral laboratories with the use of a portable, handheld potentiostat. [Copyright &y& Elsevier]

Abstract: We describe a method for linear isothermal DNA amplification using nicking endonuclease-mediated strand displacement by a DNA polymerase. The nicking of one strand of a DNA target by the endonuclease produces a primer for the polymerase to initiate synthesis. As the polymerization proceeds, the downstream strand is displaced into a single-stranded form while the nicking site is also regenerated. The combined continuous repetitive action of nicking by the endonuclease and strand-displacement synthesis by the polymerase results in linear amplification of one strand of the DNA molecule. We demonstrate that DNA templates up to 5000 nucleotides can be linearly amplified using a nicking endonuclease with 7-bp recognition sequence and Sequenase version 2.0 in the presence of single-stranded DNA binding proteins. We also show that a mixture of three templates of 500, 1000, and 5000 nucleotides in length is linearly amplified with the original molar ratios of the templates preserved. Moreover, we demonstrate that a complex library of hydrodynamically sheared genomic DNA from bacteriophage lambda can be amplified linearly. [Copyright &y& Elsevier]