Loop-mediated Isothermal Amplification Assay Research Articles

Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Two Clarireedia spp., the Causal Agent of Dollar Spot of Turfgrass.

Dollar spot is a major fungal disease affecting turfgrass worldwide and can quickly destroy turfgrass swards. An assimilating probe-based loop-mediated isothermal amplification (LAMP) assay was developed to detect Clarireedia monteithiana and C. jacksonii, the causal agents of dollar spot within the continental United States. Five LAMP primers were designed to target the calmodulin gene with the addition of a 6-carboxyl-fluorescein florescent assimilating probe, and the temperature amplification was optimized for C. jacksonii and C. monteithiana identification. The minimum amount of purified DNA needed for detection was 0.05 ng μl-1. Specificity assays against host DNA and other turfgrass pathogens were negative. Successful LAMP amplification was also observed for dollar spot-infected turfgrass field samples. Further, a DNA extraction technique via rapid heat-chill cycles and visualization of LAMP results via a florescent flashlight was developed and adapted for fast, simple, and reliable detection in 1.25 h. This assimilating probe-based LAMP assay has proved successful as a rapid, sensitive, and specific method for the detection of C. monteithiana and C. jacksonii in pure cultures and from symptomatic turfgrass leaves blades. The assay represents a promising technology to be used in the field for on-site, point-of-care pathogen detection.

Evaluation of Molecular Assays for Diagnosis of Amoebic Liver Abscess in India with Bayesian Latent Class Analysis.

Amoebic liver abscess (ALA) is the most common extra-intestinal complication of Entamoeba histolytica, accounting for 50,000 deaths annually, and is endemic in South Asia. Diagnosis based on microscopic examination is insensitive, and serological assays are not discerning of current infections in endemic settings with high exposure. For a rapid and confirmatory laboratory diagnosis of ALA, the performance of a polymerase chain reaction (PCR), quantitative real time PCR (qPCR), digital droplet PCR (ddPCR), and a loop-mediated isothermal amplification (LAMP) assay that detects E. histolytica DNA in liver abscess pus, and a lectin antigen detection ELISA were evaluated against clinical diagnosis (based on predefined criteria) as the gold standard. Owing to the lack of a laboratory gold standard, a Bayesian latent class analysis approach was also used to determine sensitivity and specificity of these assays. In the latent class analysis, qPCR and ddPCR showed the highest sensitivity (98% and 98.1%) and specificity (both 96.6%), and although clinical diagnosis had a comparable sensitivity to qPCR and ddPCR (95.2%), poorer specificity (64.3%) was seen. Kappa agreement analysis showed that qPCR and ddPCR had a perfect agreement of 1 followed by an agreement of 0.76 (95% CI: 0.64-0.88) with PCR. Considering the performance characteristics and relative ease of setting up qPCR as well as the wide availability of qPCR equipment needed, this would be the most optimal assay for rapid, confirmatory, molecular diagnosis of ALA in the tertiary care laboratory setting in India, whereas further optimization of LAMP or antibody-based detection is required for use at smaller or secondary hospitals.

Determination of limit of detection (LOD) for loop-mediated isothermal amplification (LAMP) of human cytomegalovirus (hCMV) DNA

The importance of cytomegalovirus in clinical practice remains and samples are monitored for CMV DNA titers to predict the development of disease. LAMP assays have gained increasing interest in the diagnosis of many pathogens since they do not require thermocycling, reduce the complexity of the required instrumentation as well as providing sensitivity and rapidity. So far, very few studies on CMV detection by LAMP have been reported in the literature and therefore the performance of LAMP CMV assays needs to be further characterized. In a set-up for biometrological evaluation of the suitability of the LAMP assay for CMV diagnosis, a LAMP assay was performed on a total of 192 samples with 24 replicates of 8 different hCMV DNA concentrations. The LOD was calculated as 39.09 copy/reaction (25.33 copy/reaction to 65.84 copy/reaction) with 95% confidence, representing a range that is suitable for qualitative detection. Furthermore, the lower limit of quantification was estimated at approximately 100 copy/reaction. The LOD and LLOQ values obtained in this first study to assess the biometrological relevance of LAMP CMV tests are consistent when compared to studies published before. However further study under different conditions is needed for the use of LAMP tests in clinical applications.

Serosurveillance for Plasmodium falciparum Malaria in Peruvian Army Peacekeeping Personnel, Central African Republic, 2021-2022.

Plasmodium falciparum infection threatens military populations deployed to highly malaria-endemic regions, such as Peruvian Army peacekeepers deployed to Central African Republic. During deployment, malaria cases were identified by microscopy and rapid diagnostic tests. After deployment, we performed malaria diagnosis by malachite green loop-mediated isothermal amplification and photo-induced electron transfer PCR assays. We used ELISA to test for P. falciparum C-terminal 19-kDa region merozoite surface protein 1-specific IgG from 97 peacekeepers. Malaria prevalence during deployment was 33.33% and we detected 4 cases after deployment: P. falciparum (n = 2), P. ovale (n = 1), and Plasmodium spp. (n = 1). IgG surveillance showed a seroprevalence of 31.96% in peacekeepers, who had a high P. falciparum exposure during deployment. Our findings reinforce the necessity of active surveillance in military populations to reduce the risk for introduction of new Plasmodium species and strains into the Americas from malaria-endemic areas.

Evaluation of Multiplex loop-mediated isothermal amplification assay for the detection of Mycobacterium tuberculosis complex from clinically suspected cases of pulmonary tuberculosis

Tuberculosis (TB) is the second leading cause of death from a single infectious agent worldwide. Bangladesh ranks 7th among the 30 high TB burdened countries in the world. Accurate detection of Mycobacterium tuberculosis complex (MTBC) is challenging for developing countries as most of the resource poor settings are not suitable to perform molecular techniques.The purpose of the study was to compare the multiplex TB-LAMP assay with MTB/NTM qPCR, culture, Z-N staining, and fluorescence microscopy in order to assess the effectiveness of the LAMP assay for detecting cases of pulmonary tuberculosis.This research work was done from March 2022 to February 2023. Fulfilling the inclusion criteria 130 sputum samples were collected. TB-LAMP assay, qPCR, culture in L-J media, Z-N staining and fluorescence microscopy were performed.Out of 130 samples qPCR detected MTBC in 56.92% cases and TB-LAMP detected 53.85%. MTBC was detected by culture 46.15%, by Fluorescence microscopy 40.77% and Z-N staining 36.92%. TB-LAMP detected 16.93% more cases than Z-N staining and 13.08% more cases than fluorescence microscopy. The sensitivity, specificity, positive, and negative predictive values of multiplex-LAMP assay were 95%, 81.4%, 81.4% and 95% respectively considering culture as a gold-standard. MTBC negative culture samples (18.57%) showed positivity by LAMP assay as well as by qPCR. This study detected 7.69% non-tuberculous mycobacteria (NTM) by qPCR. All NTM positive samples were negative by TB-LAMP.TB-LAMP is an easy to perform, cost-effective, reliable assay with high sensitivity and specificity. World Health Organization recommended TB-LAMP as a rapid molecular test for rapid detection of tuberculosis and as replacement of microscopy in resource poor settings/hard to reach areas. Bangladesh being a high TB burden country it is essential to implement TB-LAMP to achieve End TB Strategy by 2035.

Molecular characterization of ‘Candidatus Phytoplasma australasiaticum’ associated with ornamentals, amaranthus, French bean and sunhemp crops and development of rapid LAMP assay for detection

Phytoplasma is one of the most economically important pathogens, causing significant yield losses in many crops worldwide. During a survey in Bengaluru rural and Chikkabalpura areas of Karnataka, India, a total of 33 symptomatic plant samples including ornamentals, amaranthus, french bean, and sunhemp displaying phytoplasma symptoms were collected and confirmed as infected with phytoplasma through PCR targeting the 16S rRNA and secY genes. Pairwise sequence comparison and phylogenetic analysis of 16S rRNA and secY gene sequences confirmed the presence of ‘Candidatus Phytoplasma australasiaticum’ in antirrhinum, chrysanthemum, china aster, dianthus, amaranthus, french bean, and sunhemp. In-silico restriction fragment length polymorphism (RFLP) analysis indicated that phytoplasma strains infecting Antirrhinum, China aster, Dianthus, French bean, and Sunhemp represented distinct variants within the 16SrII phytoplasma group, with a threshold similarity coefficient of less than 0.98. A loop-mediated isothermal amplification (LAMP) assay was developed for detecting phytoplasma strains infecting the four ornamentals and other studied crops, targeting the phytoplasma 16S rRNA gene sequence with detection in 60 min. The comparative testing of LAMP products using various nucleic acid dyes and a colorimetric dye (hydroxy naphthol blue) confirmed the presence of phytoplasmas in symptomatic samples. This LAMP assay offers superior ease of use, cost-effectiveness, and rapid results for detecting phytoplasmas in symptomatic plants.

Development of point-of-need colourimetric, isothermal diagnostic assays for specific detection of Bacillus subtilis using shikimate dehydrogenase gene.

The largest obstacle in the promotion of biopesticides is the existence of counterfeit products available in the market. Identification and quantification of antagonistic organisms in biopesticide products are the key to the reduction of spurious microbial pesticides. In this study, we have developed a simple, sensitive, isothermal-based colourimetric assay for specific detection of Bacillus subtilis from the biopesticide formulations and soil samples. A region specific to B. subtilis which codes for shikimate dehydrogenase was identified through in silico analysis. We employed conventional PCR, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and qPCR for specific detection of B. subtilis in soil samples and biopesticide formulations. Specificity tests showed that the PCR primers amplified an amplicon of 521bp in four strains of B. subtilis only, and no amplification was found in negative control samples. Similarly, the LAMP assay showed sky blue colour in all four strains of B. subtilis and violet colour in negative control samples. Whereas in the RPA assay, upon the addition of SYBR Green dye, a bright green colour was seen in B. subtilis strains, while a brick-red colour was observed in negative control samples by visualizing under a UV transilluminator. The qPCR assay showed specific amplifications with a Ct value of 12 for B. subtilis strains and no amplification in negative control samples. In the sensitivity test, PCR could amplify DNA of B. subtilis up to 500pg/µL. DNA concentration as low as 10pg/µL was enough to show the colour change in the LAMP as well as the RPA assays, whereas the qPCR assay showed sensitivity till 100pg/µL. All four diagnostic assays developed in the study have been validated in soil samples and B. subtilis-based biopesticides. Compared to conventional PCR, the qPCR assay has the advantage of quantification and visualizing the result in real-time, whereas LAMP and RPA assays have the benefits of being colourimetric and less time-consuming. The other advantages are that the results can be visualized with the naked eye, and these assays do not require a costly thermal cycler and gel documentation system. Hence, LAMP and RPA assays are highly suitable for developing point-of-need diagnostic kits and, in turn, help regulators assess the quality of biopesticides in the market.

Advancements in rapid diagnostics and genotyping of Piscirickettsia salmonis using Loop-mediated Isothermal Amplification.

Piscirickettsia salmonis, the causative agent of Piscirickettsiosis, poses a significant threat to the Chilean aquaculture industry, resulting in substantial economic losses annually. The pathogen, first identified as specie in 1992, this pathogen was divided into two genogroups: LF-89 and EM-90, associated with different phenotypic mortality and pathogenicity. Traditional genotyping methods, such as multiplex PCR, are effective but limited by their cost, equipment requirements, and the need for specialized expertise. This study validates Loop-mediated Isothermal Amplification (LAMP) as a rapid and specific alternative for diagnosing P. salmonis infections. We developed the first qPCR and LAMP assay targeting the species-conserved tonB receptor gene (tonB-r, WP_016210144.1) for the specific species-level identification of P. salmonis. Additionally, we designed two genotyping LAMP assays to differentiate between the LF-89 and EM-90 genogroups, utilizing the unique coding sequences Nitronate monooxygenase (WP_144420689.1) for LF-89 and Acid phosphatase (WP_016210154.1) for EM-90. The LAMP assays demonstrated sensitivity and specificity comparable to real-time PCR, with additional benefits including rapid results, lower costs, and simplified operation, making them particularly suitable for field use. Specificity was confirmed by testing against other salmonid pathogens, such as Renibacterium salmoninarum, Vibrio ordalii, Flavobacterium psychrophilum, Tenacibaculum maritimum, and Aeromonas salmonicida, with no cross-reactivity observed. The visual detection method and precise differentiation between genogroups underscore LAMP's potential as a robust diagnostic tool for aquaculture. This advancement in the specie detection (qPCR and LAMP) and genotyping of P. salmonis represents a significant step forward in disease management within the aquaculture industry. The implementation of LAMP promises enhanced disease surveillance, early detection, and improved management strategies, ultimately benefiting the salmonid aquaculture sector.

Development and Validation of a Highly Sensitive RT-qLAMP Assay for Rapid Detection of SARS-CoV-2: Methodological Aspects.

Specific and reliable diagnostic methods are becoming increasingly essential to identify patients in light of the high transmission rate and the recent appearance of the new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For the specific detection of SARS-CoV-2, our quantitative reverse transcription loop-mediated isothermal amplification (RT-qLAMP) assay implementation demonstrates how flexible it can be with two readouts: visualized colorimetric and real-time fluorescence. Different factors were optimized to improve the reaction conditions, including temperature (60°C), assay runtime (60min), primers, MgSO4 (6mM), dNTPs (1mM), LAMP Buffer (1.2mM Tris-HCl), KCl (50mM), pH (8), and phenol red (10mM) concentrations. Regarding analytical sensitivity, the colorimetric RT-LAMP method detected samples with Ct values up to 29, while the RT-qLAMP assay identified up to Ct = 31. RT-qLAMP was evaluated on 40 clinical samples (25 positives and 15 negatives) for viral RNA detection. All negative samples were found negative through fluorescent reading in RT-qLAMP and quantitative reverse transcription PCR (RT-qPCR) assays. Twenty-three clinically positive samples demonstrated a positive RT-qLAMP reaction (up to Ct ≤ 31) with 92% clinical sensitivity, 100% clinical specificity, 100% positive predictive value (PPV), 88.24% negative predictive values (NPV), and 95% accuracy.

Optimization of Reverse Transcription Loop-Mediated Isothermal Amplification for In Situ Detection of SARS-CoV-2 in a Micro-Air-Filtration Device Format.

The Coronavirus disease 2019 (COVID-19) pandemic has supercharged innovation in the field of molecular diagnostics and led to the exploration of systems that permit the autonomous identification of airborne infectious agents. Airborne virus detection is an emerging approach for determining exposure risk, although current methods limit intervention timeliness. Here, we explore reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for one-pot detection of Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) (SCV2) run on membrane filters suitable for micro-air-filtration of airborne viruses. We use a design of experiments statistical framework to establish the optimal additive composition for running RT-LAMP on membrane filters. Using SCV2 liquid spike-in experiments and fluorescence detection, we show that single-pot RT-LAMP on glass fiber filters reliably detected 0.10 50% tissue culture infectious dose (TCID50) SCV2 per reaction (3600 E-gene copies) and is an order of magnitude more sensitive than conventional RT-LAMP.

Development and Validation of the MAST ISOPLEX®VTEC Kit for Simultaneous Detection of Shiga Toxin/Verotoxin 1 and 2 (stx1/vt1 and stx2/vt2) with Inhibition Control (IC) in a Rapid Loop-Mediated Isothermal Amplification (LAMP) Multiplex Assay.

Loop-mediated isothermal amplification (LAMP) is a cost-effective, rapid, and highly specific method of replicating nucleic acids. Adding multiple targets into a single LAMP assay to create a multiplex format is highly desirable for clinical applications but has been challenging due to a need to develop specific detection techniques and strict primer design criteria. This study describes the evaluation of a rapid triplex LAMP assay, MAST ISOPLEX®VTEC, for the simultaneous detection of Shiga toxin/verotoxin 1 and 2 (stx1/vt1 and stx2/vt2) genes in verotoxigenic Escherichia coli (E. coli) (VTEC) isolates with inhibition control (IC) synthetic DNA using a single fluorophore-oligonucleotide probe, MAST ISOPLEX®Probes, integrated into the primer set of each target. MAST ISOPLEX®Probes used in the MAST ISOPLEX®VTEC kit produce fluorescent signals as they integrate with reaction products specific to each target, allowing tracking of multiple amplifications in real time using a real-time analyzer. Initial validation on DNA extracts from fecal cultures and synthetic DNA sequences (gBlocks) showed that the MAST ISOPLEX®VTEC kit provides a method for sensitive simultaneous triplex detection in a single assay with a limit of detection (LOD) of less than 100 target copies/assay and 96% and 100% sensitivity and specificity, respectively.

A simple color absorption analysis of colorimetric loop-mediated isothermal amplification for detection of Raillietina spp. in clinical samples using a 3D-printed tube holder coupled with a smartphone camera and notebook screen.

Asimple method has been developedfor semi-quantitative analysis of the colorimetric output of loop-mediated isothermal amplification (LAMP) using a 3D-printed tube holder with a smartphone and notebook for the detection of Raillietina, which is the cause of Raillietiniasis affecting free-range chicken farming. In this method, a light is directedfrom a notebook screen to the LAMP products in the tube holder and the color absorption of the LAMP products is measured by using the appropriate smartphone application. It was found that the malachite green dye-coupled LAMP (MaG-LAMP) assay showed the highest sensitivity and specificity for detecting Raillietina without any cross-reaction with other related parasites and hosts. The limit of detection was 10fg/μL of DNA. A total of 60 fecal samples were infectively confirmed by microscopic examination and the results of microscopy compared with those of MaG-LAMP and triplex PCR assays. Microscopy and MaG-LAMP based on the color absorption demonstrated high agreement in Raillietina detection with kappa = 1. Rapid, simple, cost-effective, and easy interpretation of colorimetric LAMP assays and their high sensitivity make them superior to PCR and morphological investigation, demonstrating the feasibility of this assay in point-of-care screening to support farm management and solve chicken health problems. Our study presentsis an alternative diagnostic method using semi-quantitative analysis of colorimetric LAMP based on the differing solution color absorptions between positive and negative reactions for infectious disease diagnosis.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) method for Candida glabrata detection.

Loop-mediated isothermal amplification (LAMP) is a simple and rapid nucleic acid method for DNA amplification at a constant temperature. The "gold standard" culture method for yeast detection, has low sensitivity with severe consequences, increasing morbidity and mortality rates. Here, we report the development of a LAMP method for the specific detection of C. glabrata. The specific LAMP primers for C. glabrata detection were designed and evaluated. The LAMP assay accurately detected C. glabrata with no cross-reactivity with other Candida species. The developed molecular method would be a promising tool in the management of invasive candidiasis.

Aptamer-based Strategies for COVID-19 Detection and Treatment: A Systematic Review Study

Background: Aptamer-based strategies have emerged as promising tools for the detection and treatment of COVID-19, offering advantages such as high specificity, sensitivity, and versatility. This systematic review aims to evaluate the effectiveness and innovation of aptamer-based approaches for COVID-19 detection and treatment. Methods: Following the guidelines of the Cochrane Handbook for Systematic Reviews and the PRISMA 2020 guidelines, a systematic search was conducted across multiple databases up to 2024. The search included studies that utilized aptamers for the diagnosis or therapy of COVID-19. Screening and selection of studies were performed independently by two reviewers, with any disagreements resolved by a third reviewer. Data were extracted regarding study characteristics, aptamer details, and outcomes. Results: In our systematic review, 98 studies from an initial pool of 1541 records met the inclusion criteria for analysis. Aptamers, single-stranded DNA or RNA molecules with unique threedimensional (3D) structures, were extensively explored for COVID-19 detection and treatment. Various aptamer-based assays, including electrochemical sensors, surface plasmon resonance (SPR) biosensors, and lateral flow assays, demonstrated high sensitivity and specificity in detecting SARSCoV- 2 in clinical samples such as saliva, nasal swabs, and wastewater. Several aptamer structures targeting viral proteins like the spike and nucleocapsid proteins were employed. Nucleic Acid Amplification Techniques (NAATs) utilizing aptamers, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based and Loop-mediated Isothermal Amplification (LAMP) assays, showed exceptional sensitivity in detecting viral genetic material. Aptamer-based therapeutic approaches showed potential by blocking viral protein activity or serving as delivery vehicles for therapeutic agents like small interfering RNAs (siRNAs). Despite their advantages, aptamer technologies face limitations such as susceptibility to nuclease degradation and rapid renal clearance, highlighting the need for further optimization. Conclusion: Aptamer-based strategies present promising avenues for COVID-19 detection and treatment. These approaches offer advantages such as high sensitivity, specificity, and rapid detection, making them valuable tools in combating the COVID-19 pandemic. Further research and development are warranted to optimize aptamer-based strategies for widespread application in clinical settings.

Loop-mediated isothermal amplification (LAMP) for detection of atypical enterovirus D68 strain VR-1197

A method that has rapidly evolved for detection of viral pathogens are loop-mediated isothermal amplification (LAMP) assays. The available LAMP assays usually target the most common viral strains, including enteroviruses, but for the atypical enterovirus D68 strain VR-1197 this method has not yet been developed. Enterovirus D68 are known for severe respiratory distress in children, and atypical strains are less likely to be detected by traditional methods. This study targets the atypical EVD68 strain VR-1197 and have developed a rapid detection method saving time when differentiating enterovirus strains. This study present method development and review the sensitivity and specificity compared to traditional RT-qPCR, and wet lab cross reactivity with other airway pathogens. The EVD68 VR-1197 assay can be a rapid POC (Point of care) test for atypical EVD68 VR-1197 and have the potential as reliable detection method with minimal technological requirements.

Loop-Mediated Isothermal Amplification Assay to Detect Invasive Malaria Vector Anopheles stephensi Mosquitoes.

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.

Sex identification of sun conure (Aratinga solstitialis) using loop-mediated isothermal amplification of W and Z spindlin chromosomes.

The sun conure (Aratinga solstitialis), a bird belonging to the Psittaciformes family, is a popular pet because of its bright color and beautiful appearance. The sun conure is a monomorphic bird with similar appearances between males and females, making sex identification difficult by observing the external morphology. Therefore, molecular techniques are utilized. Loop-mediated isothermal amplification (LAMP) is a molecular technique that is often applied for sex identification in birds and is a quick and simple method that can be used in the field. This study used the LAMP technique to improve sex identification in sun conures by observing the color change of hydroxy naphthol blue. Two primer sets, SunSpin-W and SunSpin-Z, were designed for sex identification in sun conures using the LAMP technique specific to the spindlin gene. The developed LAMP reaction was tested for optimal conditions, sensitivity, and specificity compared with the polymerase chain reaction (PCR) technique. The SunSpin-W primer set amplified only female birds, whereas the SunSpin-Z primer set amplified DNA from both male and female birds. The primer sets were optimized at 62°C for 45 min. A positive result was visible to the naked eye from the color change of the reaction. In the LAMP assay, the lowest detectable concentration was 10 pg/μL and in the PCR assay, it was 1 ng/μL, while a 100% accuracy rate in sex identification was observed when comparing the LAMP assay results with the PCR assay. This study successfully developed a LAMP technique for sex identification of sun conure, which took 45 min to complete and can be expanded for use in the field.

Rapid and Cost‐Effective Platypus eDNA Detection in Waterways Using Loop‐Mediated Isothermal Amplification Assay: Advancing Conservation Efforts

ABSTRACTFreshwater ecosystems, home to a remarkable diversity of species, are facing severe threats from human activities such as climate change, habitat degradation, over‐extraction of water for irrigation, and pollution. The platypus, an iconic species in freshwater ecosystems around Australia, is threatened by all these activities, both singly and in combination. The scale and complexity of these intersecting and reinforcing threats makes cost‐effective monitoring tools essential to better understand how platypus populations are responding. In this study, we optimized a loop‐mediated isothermal amplification (LAMP) assay for the rapid and cost‐effective detection of platypus DNA in environmental water samples, offering an attractive alternative to quantitative polymerase chain reaction (qPCR). We improved a water filtration protocol for in‐field use, employing suitable filter membranes for processing large volumes of water, thereby maximizing DNA recovery from dilute samples. The limit of detection for the platypus LAMP (Plat‐LAMP) assay was determined to be 12.4 copies/μL using a standard plasmid positive reference and 7 × 10−6 ng/μL when applied to DNA extracted from platypus tissue, with improved sensitivity achieved through the incorporation of locked nucleic acid primers. In comparative testing against qPCR, the Plat‐LAMP assay exhibited greater sensitivity in detecting platypus DNA in known positive water samples collected from platypus habitat at Healesville Sanctuary. Furthermore, the Plat‐LAMP assay demonstrated 100% specificity when performed on water samples collected from non‐platypus habitats. In field testing across waterways in Victoria and New South Wales, the Plat‐LAMP assay detected platypus in 36.96% of samples, compared to 54.35% of samples using qPCR. These findings underscore the Plat‐LAMP assay's potential as a faster and more cost‐effective complementary method to qPCR, rendering it suitable for point‐of‐application water testing. The ability to conduct eDNA surveys without the need for cold‐chain logistics would significantly assist conservation organizations and water managers map platypus distributions and facilitate conservation efforts around Australia.

Interlaboratory Harmonization Study and Prospective Evaluation of the PURE–Trypanosoma cruzi–Loop-Mediated Isothermal Amplification Assay for Detecting Parasite DNA in Newborn's Dried Blood Spots

Timely diagnosis of vertical Trypanosoma cruzi infections involves microscopy-based detection of circulating parasites from peripheral blood, which lacks sensitivity and is operator dependent. Consequently, most children born to T. cruzi-infected mothers are required to undergo serological testing after nine months, which risks loss to follow-up. Alternatively, the Loop-mediated isothermal amplification (LAMP) test for T. cruzi DNA offers high analytical and clinical performance and easy to use in low-complexity laboratories. Recently, we optimized this technique using an ultra-rapid DNA extraction method combined with the LAMP in dried blood spots (DBS) on FTA cards. The procedure has been implemented in ten public maternities across Paraguay, Bolivia, and Argentina, involving the training of 14 technicians. Operators' performance was evaluated using a standardized DBS testing panel for harmonization, including negative controls and DBS samples artificially contaminated with T. cruzi at 50 and 20 cells/mL. There was strong agreement (ĸ = 0.924) for controls and 50 cells/mL samples, and good agreement (ĸ = 0.718) across all testing panels, even at the detection limit of the test. A prospective study collected 306 DBS from 222 newborns at birth and/or two months, detecting T. cruzi microscopically in four cases. LAMP identified eight positive cases and perfectly aligned with Real Time PCR (ĸ = 1), demonstrating higher sensitivity than microscopic observation for early detection of infection in infants.

Development of a highly specific LAMP assay for detection of Sarcocystis tenella and Sarcocystis gigantea in sheep.

Sarcocystis infection in sheep has caused significant economic losses in the livestock industry, and the genetic similarity among Sarcocystis species highlights the need for precise diagnostic methods in sheep. This study developed a loop-mediated isothermal amplification (LAMP) method targeting COX-1 and 28S rRNA genes to detect Sarcocystis tenella and Sarcocystis gigantea, respectively. The LAMP method exhibited high specificity, selectively amplifying target DNA sequences without cross-reactivity with closely related protozoa, such as Toxoplasma gondii and Neospora caninum. Detection limits were determined as 3 × 105 copies/L for S. tenella and 6 × 104 copies/L for S. gigantea, enabling sensitive identification of low-level infections. Comparative analysis with conventional PCR on sheep cardiac tissues demonstrated a higher LAMP detection rate (80.0% vs 66.7%). In conclusion, the LAMP method offers superior sensitivity to conventional PCR, allows visual confirmation of results, and provides a rapid diagnostic tool for identifying S. tenella and S. gigantea infection in sheep. However, due to the limitation of sample availability, we were unable to assess all Sarcocystis species that use sheep as intermediate hosts, which warrants further research.