Summary
The novel coronavirus disease‐2019 (Covid‐19) public health emergency has caused enormous loss around the world. This pandemic is a concrete example of the existing gap between availability ...of advanced diagnostics and current need for cost‐effective methodology. The advent of the loop‐mediated isothermal amplification (LAMP) assay provided an innovative tool for establishing a rapid diagnostic technique based on the molecular amplification of pathogen RNA or DNA. In this review, we explore the applications, diagnostic effectiveness of LAMP test for molecular diagnosis and surveillance of severe acute respiratory syndrome coronavirus 2. Our results show that LAMP can be considered as an effective point‐of‐care test for the diagnosis of Covid‐19 in endemic areas, especially for low‐ and middle‐income countries.
Spread of the Anopheles stephensi mosquito, an invasive malaria vector, threatens to put an additional 126 million persons per year in Africa at risk for malaria. To accelerate the early detection ...and rapid response to this mosquito species, confirming its presence and geographic extent is critical. However, existing molecular species assays require specialized laboratory equipment, interpretation, and sequencing confirmation. We developed and optimized a colorimetric rapid loop-mediated isothermal amplification assay for molecular An. stephensi species identification. The assay requires only a heat source and reagents and can be used with or without DNA extraction, resulting in positive color change in 30-35 minutes. We validated the assay against existing PCR techniques and found 100% specificity and analytical sensitivity down to 0.0003 ng of genomic DNA. The assay can successfully amplify single mosquito legs. Initial testing on samples from Marsabit, Kenya, illustrate its potential as an early vector detection and malaria mitigation tool.Spread of the Anopheles stephensi mosquito, an invasive malaria vector, threatens to put an additional 126 million persons per year in Africa at risk for malaria. To accelerate the early detection and rapid response to this mosquito species, confirming its presence and geographic extent is critical. However, existing molecular species assays require specialized laboratory equipment, interpretation, and sequencing confirmation. We developed and optimized a colorimetric rapid loop-mediated isothermal amplification assay for molecular An. stephensi species identification. The assay requires only a heat source and reagents and can be used with or without DNA extraction, resulting in positive color change in 30-35 minutes. We validated the assay against existing PCR techniques and found 100% specificity and analytical sensitivity down to 0.0003 ng of genomic DNA. The assay can successfully amplify single mosquito legs. Initial testing on samples from Marsabit, Kenya, illustrate its potential as an early vector detection and malaria mitigation tool.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Because of the serological cross-reactivity among the flaviviruses, molecular detection methods, such as reverse-transcription polymerase chain reaction (RT-PCR), play an important role in the recent ...Zika outbreak. However, due to the limited sensitivity, the detection window of RT-PCR for Zika viremia is only about one week after symptom onset. By combining loop-mediated isothermal amplification (LAMP) and AC susceptometry, we demonstrate a rapid and homogeneous detection system for the Zika virus oligonucleotide. Streptavidin-magnetic nanoparticles (streptavidin-MNPs) are premixed with LAMP reagents including the analyte and biotinylated primers, and their hydrodynamic volumes are dramatically increased after a successful LAMP reaction. Analyzed by a portable AC susceptometer, the changes of the hydrodynamic volume are probed as Brownian relaxation frequency shifts, which can be used to quantify the Zika virus oligonucleotide. The proposed detection system can recognize 1 aM synthetic Zika virus oligonucleotide in 20% serum with a total assay time of 27min, which can hopefully widen the detection window for Zika viremia and is therefore promising in worldwide Zika fever control.
•Synthetic Zika virus oligonucleotide is quantified by an AC susceptometer.•Amplicons of the LAMP reaction are attached to the magnetic nanoparticles.•Mg2P2O7 deposits onto the nanoparticles and further improves the signal.•An LOD of 1 aM oligos can be achieved with a total assay time of 27min.
Loop-mediated isothermal amplification (LAMP), a newly developed gene amplification method, combines rapidity, simplicity, and high specificity. Several tests have been developed based on this ...method, and simplicity is maintained throughout all steps, from extraction of nucleic acids to detection of amplification. In the LAMP reaction, samples are amplified at a fixed temperature through a repetition of two types of elongation reactions occurring at the loop regions: self-elongation of templates from the stem loop structure formed at the 3′-terminal and the binding and elongation of new primers to the loop region. The LAMP reaction has a wide range of possible applications, including point-of-care testing, genetic testing in resource-poor settings (such as in developing countries), and rapid testing of food products and environmental samples.
Digital loop‐mediated isothermal amplification (dLAMP) refers to compartmentalizing nucleic acids and LAMP reagents into a large number of individual partitions, such as microchambers and droplets. ...This compartmentalization enables dLAMP to be an excellent platform to quantify the absolute number of the target nucleic acids. Owing to its low requirement for instrumentation complexity, high specificity, and strong tolerance to inhibitors in the nucleic acid samples, dLAMP has been recognized as a simple and accurate technique to quantify pathogenic nucleic acid. Herein, the general process of dLAMP techniques is summarized, the current dLAMP techniques are categorized, and a comprehensive discussion on different types of dLAMP techniques is presented. Also, the challenges of the current dLAMP are illustrated together with the possible strategies to address these challenges. In the end, the future directions of the dLAMP developments, including multitarget detection, multisample detection, and processing nucleic acid extraction are outlined. With recently significant advances in dLAMP, this technology has the potential to see more widespread use beyond the laboratory in the future.
Herein, the concept of digital loop mediated isothermal amplification (dLAMP), which allows quantification of the absolute number of nucleic acids, is presented. The current dLAMP techniques, including their advantages and disadvantages are summarized. Furthermore, the challenges faced when applying dLAMP are discussed together with their potential solutions, followed by an outline of their future developments.
Listeria monocytogenes (LMO) represents one of the most serious hazards for food health due to the high mortality rate and its ability to survive at limiting conditions. In this study, an ...electrochemical sensor for the detection of LMO in food samples using loop mediated isothermal amplification (LAMP) technique is presented. The extraction step for the release of the nucleic acids of the sample was simplified using three different alternatives (QuickExtract buffer lysis, heating method and an extraction-free option). The three protocols were tested with the LAMP reaction in seven different food matrices (Dairy milk, fresh cheese, ham, hummus, lettuce, milk powder and pâté) and were able to detect 1 cfu/25 g as stipulated by the food safety regulations. It was proved that all three alternatives were compatible with the electrochemical detection using methylene blue redox-active molecule in dairy milk samples. The final validation of the sensor was carried out with the extraction-free option in nine different food matrices, which were able to detect 1 cfu/25 g of food sample in all cases. This validation demonstrates the feasibility of the sensor for its application in the food industry as a point-of-test solution.
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•Electrochemical sensor for the detection of Listeria monocytogenes by LAMP.•The limit of detection of the LAMP reaction is 312 fg of DNA per reaction.•The electrochemical detection is compatible with an extraction-free amplification.•The EC-LAMP sensor detects 1 cfu of Listeria monocytogenes in 25 g of food sample.
Plant viruses cause considerable economic losses and are a threat for sustainable agriculture. The frequent emergence of new viral diseases is mainly due to international trade, climate change, and ...the ability of viruses for rapid evolution. Disease control is based on two strategies: i) immunization (genetic resistance obtained by plant breeding, plant transformation, cross-protection, or others), and ii) prophylaxis to restrain virus dispersion (using quarantine, certification, removal of infected plants, control of natural vectors, or other procedures). Disease management relies strongly on a fast and accurate identification of the causal agent. For known viruses, diagnosis consists in assigning a virus infecting a plant sample to a group of viruses sharing common characteristics, which is usually referred to as species. However, the specificity of diagnosis can also reach higher taxonomic levels, as genus or family, or lower levels, as strain or variant. Diagnostic procedures must be optimized for accuracy by detecting the maximum number of members within the group (sensitivity as the true positive rate) and distinguishing them from outgroup viruses (specificity as the true negative rate). This requires information on the genetic relationships within-group and with members of other groups. The influence of the genetic diversity of virus populations in diagnosis and disease management is well documented, but information on how to integrate the genetic diversity in the detection methods is still scarce. Here we review the techniques used for plant virus diagnosis and disease control, including characteristics such as accuracy, detection level, multiplexing, quantification, portability, and designability. The effect of genetic diversity and evolution of plant viruses in the design and performance of some detection and disease control techniques are also discussed. High-throughput or next-generation sequencing provides broad-spectrum and accurate identification of viruses enabling multiplex detection, quantification, and the discovery of new viruses. Likely, this technique will be the future standard in diagnostics as its cost will be dropping and becoming more affordable.
Enzymatic DNA amplification‐based approaches involving intercalating DNA‐binding fluorescent dyes and expensive optical detectors are the gold standard for nucleic acid detection. As components of a ...simplified and miniaturized system, conventional silicon‐based ion sensitive field effect transistors (ISFETs) that measure a decrease in pH due to the generation of pyrophosphates during DNA amplification have been previously reported. In this article, Bst polymerase in a loop‐mediated isothermal amplification (LAMP) reaction combined with target‐specific primers and crumpled graphene field effect transistors (gFETs) to electrically detect amplification by sensing the reduction in primers is used. Graphene is known to adsorb single‐stranded DNA due to noncovalent π–π bonds, but not double‐stranded DNA. This approach does not require any surface functionalization and allows the detection of primer concentrations at the endpoint of reactions. As recently demonstrated, the crumpled gFET over the conventional flat gFET sensors due to their superior sensitivity is chosen. The endpoint of amplification reaction with starting concentrations down to 8 × 10−21 m in 90 min including the time of amplification and detection is detected. With its high sensitivity and small footprint, this platform will help bring complex lab‐based diagnostic and genotyping amplification assays to the point‐of‐care.
Selective adsorption of only single‐stranded DNA molecules on graphene is used to detect enzymatic DNA amplification by sensing the consumption of single‐stranded DNA primers in an isothermal amplification reaction.
The technique of loop-mediated isothermal amplification (LAMP) utilizes 4 (or 6) primers targeting 6 (or 8) regions within a fairly small segment of a genome for amplification, with concentration ...higher than that used in traditional PCR methods. The high concentrations of primers used leads to an increased likelihood of non-specific amplification induced by primer dimers. In this study, a set of LAMP primers were designed targeting the prfA gene sequence of Listeria monocytogenes, and dimethyl sulfoxide (DMSO) as well as Touchdown LAMP were employed to increase the sensitivity and specificity of the LAMP reactions. The results indicate that the detection limit of this novel LAMP assay with the newly designed primers and additives was 10 fg per reaction, which is ten-fold more sensitive than a commercial Isothermal Amplification Kit and hundred-fold more sensitive than previously reported LAMP assays. This highly sensitive LAMP assay has been shown to detect 11 strains of Listeria monocytogenes, and does not detect other Listeria species (including Listeria innocua and Listeria invanovii), providing some advantages in specificity over commercial Isothermal Amplification Kits and previously reported LAMP assays by Tang, et al.
Background
The current methods for detecting Mycobacterium tuberculosis (Mtb) are not clinically optimal. Standard culture methods (SCMs) are slow, costly, or unreliable, and loop‐mediated isothermal ...amplification (LAMP) cannot differentiate live Mtb.
Methods
This study compared reverse transcription (RT)‐LAMP, LAMP, and an SCM for detecting Mtb. A first experiment tested the sensitivity and specificity of primers for 9 species of Mycobacterium (H37Rv, M. intracellulare, M. marinum, M. kansasii, M. avium, M. flavescens, M. smegmatis, M. fortuitum, and M. chelonae); and 3 non‐Mycobacterium species (Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae). A second experiment tested sputum specimens for the presence of Mtb, from 100 patients with tuberculosis (clinical) and 22 from patients without tuberculosis (control), using Roche solid culture (SCM), LAMP, and RT‐LAMP. In the clinical samples.
Results
The rates of positivity for Mtb of the SCM, LAMP, and RT‐LAMP methods were 88%, 92%, and 100%, respectively. The difference in detection rate was significant between RT‐LAMP and SCM, but RT‐LAMP and LAMP were comparable. In the control group, the detection rates were nil for all three methods.
Conclusion
The specificities of the methods were similar. The sensitivity of RT‐LAMP was ~10‐fold higher than that of LAMP for detecting Mtb. Unlike LAMP, RT‐LAMP could identify viable bacteria, and was able to detect a single copy of Mtb. Among SCM, LAMP, and RT‐LAMP, the latter is the most suitable for wide use in the lower‐level hospitals and clinics of China for detecting Mtb in sputum samples.
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