Development Of Loop-mediated Isothermal Amplification Assay Research Articles

Rapid on-site authentication of three cockle species using real-time loop-mediated isothermal amplification (LAMP) assay

Cockles, crucial for commercial fishing and aquaculture, emerge as highly marketable shellfish. However, the morphological similarities among various cockle species are frequently exploited for shellfish fraud. In this study, we developed LAMP assays (end-point LAMP and real-time LAMP) to detect three commercially important cockle species: Tegillarca granosa, Anadara broughtonii, and Anadara kagoshimensis. The primers were designed based on mitochondrial DNA and used to develop LAMP assays. Exclusivity was assessed using 17 shellfish species, and no cross-reactivity was confirmed. The estimate of the detection limit was determined to be 0.0001 ng. The established assays were further refined to create an on-site real-time LAMP assay by combining fast DNA isolation with a real-time fluorometer for field application. The effectiveness of the on-site real-time LAMP assay was successfully verified using 38 commercial samples, with analysis accuracy confirmed using a DNA extraction kit. Consequently, the on-site real-time LAMP assay can complete the whole procedure, including DNA extraction, real-time LAMP reaction, and data analysis within a 45 min timeframe, maintaining the accuracy of detecting three cockle species from processed cockle samples in the field. Our method proves valuable for detecting shellfish fraud by identifying cockle species using samples of both raw and processed products in the field.

Rapid and visual detection of Shiga-toxigenic Escherichia coli (STEC) in carabeef meat harnessing loop-mediated isothermal amplification (LAMP).

Shiga toxigenic E. coli are important foodborne zoonotic pathogens. The present study was envisaged to standardize loop-mediated isothermal amplification assays targeting stx1 and stx2 genes for rapid and visual detection of STEC and compare its sensitivity with PCR. The study also assessed the effect of short enrichment on the detection limit of LAMP and PCR. The developed LAMP assays were found to be highly specific. Analytical sensitivity of LAMP was 94fg/µLand 25.8fg/µL for stx-1 and stx-2 while LOD of 5 CFU/g of carabeef was measured after 6-12h enrichment. The study highlights the importance of short (6-12h) enrichment for improving the sensitivity of LAMP. The entire detection protocol could be performed within 9h yielding results on the same day. The developed LAMP assays proved to be a handy and cost-effective alternative for screening STEC contamination in meat.

Development of a colorimetric loop‐mediated isothermal amplification (LAMP) assay to facilitate monitoring of carbendazim‐resistant isolates of Colletotrichum truncatum based on the β‐tubulin E198A mutation

AbstractAnthracnose, a disease mainly caused by Colletotrichum truncatum, is a serious threat to vegetable soybean production worldwide. Carbendazim, a methyl‐benzimidazole carbamate fungicide, has been frequently applied to control the disease. Unfortunately, carbendazim‐resistant isolates of C. truncatum have emerged, resulting in inefficient disease control. Therefore, a rapid and simple method for monitoring the fungicide‐resistant isolates in fungal populations is essential to select the most appropriate strategy to control the disease and to handle fungicide resistance in a timely manner. In this study, we developed a colorimetric loop‐mediated isothermal amplification (LAMP) assay to detect carbendazim‐resistant isolates by using the E198A mutation in the TUB2 gene as the marker. With the optimal primers, the developed LAMP assay could detect resistant isolates after incubation for 40 min at 67°C. The results could be simply observed with the naked eye, and the assay provided 10‐fold higher sensitivity than conventional PCR. Moreover, the specificity, accuracy, and reproducibility of the generated LAMP were shown in testing with 85 C. truncatum isolates from five locations in northern Thailand. Resistant isolates carrying the F200Y mutation were not detected with the LAMP assay. Thus, the developed colorimetric LAMP assay could be used as a rapid and simple detector of certain carbendazim‐resistant isolates of C. truncatum.

Fast and User-Friendly Detection of Flatfish Species (Pleuronectes platessa and Solea solea) via Loop-Mediated Isothermal Amplification (LAMP).

The detection of a Cytochrome b gene (cytb) for species differentiation in fish is intensively used. A fast alternative to expensive and time-consuming DNA barcoding is loop-mediated isothermal amplification (LAMP) in combination with efficient readout systems. For this reason, we developed LAMP assays for rapid species detection of Pleuronectes platessa and Solea solea, two economically important flatfish species in Europe that are prone to mislabeling. Species-specific primer sets targeting cytb were designed, and LAMP assays were optimized. With the optimized LAMP assays, we were able to detect up to 0.1 and 0.01 ng of target DNA of P. platessa and S. solea, respectively, and in each case up to 1% (w/w) of target species in mixtures with nontarget species. For future on-site detection, a lateral flow assay and a pocket-sized lab-on-phone assay were used as readout systems. The lab-on-phone assay with the S. solea specific primer set revealed cross-reactivity to Solea senegalensis. The assay targeting P. platessa proved to be highly specific. Both assays could be performed within 45 min and provided rapid and easy detection of fish species.

A loop-mediated isothermal amplification assay for the rapid diagnosis of soybean rust caused by Phakopsora pachyrhizi

Soybean rust caused by the fungus Phakopsora pachyrhizi is one of the most destructive diseases to soybean production worldwide. For the rapid diagnosis and the control of soybean rust, we developed a loop-mediated isothermal amplification (LAMP) assay that targets a P. pachyrhizi-specific gene (Phapa_6409908) identified from a comparative genomic analysis of 61 Pucciniomycotina strains. Using a set of screened primers and the optimized reaction conditions of 62°C for 70 min, the LAMP assay can detect P. pachyrhizi while excluding other plant pathogens. The assay consistently showed high sensitivity in detecting low contents of P. pachyrhizi DNA (10 pg). We confirmed the practical application of this LAMP assay in diagnosing soybean rust using soybean leaf samples collected from fields in four cities of Sichuan Province, China, in 2020 and 2021. The developed LAMP assay provides a specific, sensitive, and rapid method for the diagnosis of soybean rust caused by P. pachyrhizi, which can assist in the prediction, prevention, and control of this major soybean disease.

Detection of post-harvest pathogens by loop-mediated isothermal amplification: a review

Postharvest losses, which occur between harvest and consumption of agricultural commodities, are major causes of food waste. Minimizing food loss helps provide nutritious food for animals and humans, and alleviate adverse environmental effects on food production. These losses are often related to the presence of postharvest pathogens, including fungi and bacteria, which typically start by infecting crops in the field as well as during postharvest chain. Control of these pathogens relies on development of tools that ensure their early and accurate detection. Among these is loop-mediated isothermal amplification (LAMP), a molecular method for pathogen detection. LAMP characteristics of rapidity, specificity and simplicity have encouraged development of a number of LAMP assays for detection of postharvest pathogens. Each LAMP assay allows to detect a specific genetic region of the target microorganism, which can be directly related to mycotoxin production, fungicide resistance and phytotoxicity. The LAMP amplicons are rapidly visualized, either at a specific timepoint, or in real-time by taking measurements throughout reaction, thereby necessitating less sophisticated facilities than those needed for PCR assays. In addition, many studies have developed simple protocols for the direct detection of pathogens on fresh produce. This paper explains the LAMP reaction, and its importance for postharvest detection of fungi and bacteria. Previous studies that have developed LAMP assays are also discussed.

Rapid and simple colorimetric detection of quiescent Colletotrichum in harvested fruit using reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) technology

Anthracnose, caused by the fungus Colletotrichum gloeosporioides, is one of the major causes of postharvest decay of fruits and vegetables. Detection of the pathogen at an early stage of infection is crucial to developing a disease management strategy. In this work, a loop-mediated isothermal amplification (LAMP) assay was developed for the rapid detection of C. gloeosporioides targeting the transcript enoyl-CoA hydratase (ECH) that significantly upregulates only during C. gloeosporioides quiescent stage. The assay enabled a naked-eye detection of C. gloeosporioides RNA within 23 min based on a color change of LAMP products from pink to yellow. The detection limit of the LAMP assay was 1 pg of total RNA extracted from fruit peel in a 25 μL reaction. Positive results were obtained only in samples carrying the ECH gene, whereas no cross-reaction was observed for a different quiescent marker (histone deacetylase (HDAC)) or an appressorium marker (scytalone dehydratase, (SD)), indicating the high specificity of the method. Hence, the results indicate that the developed LAMP assay is a rapid, highly sensitive, and specific tool for the early detection of quiescent C. gloeosporioides and could be employed to manage postharvest diseases.

Development of rapid and sensitive loop-mediated isothermal method for on-site visual identification of tissue origin of pig by using mitochondrial COI gene sequences

Context The loop-mediated isothermal amplification (LAMP) methods have great potential to identify the species origin of the tissue in meat and meat products at isothermal temperature and are also suitable for field conditions. Aim The present study aimed to develop a rapid, specific, and sensitive assay based on the LAMP technique for identification of tissue of pig origin. Methods The pig-specific primers were designed by targeting the mitochondrial COI gene. The amplification temperature and time for the LAMP reaction were optimised as 64°C and 45 min. The analysis of the amplified product was performed on the basis of the development of colour after the addition of intercalating SYBR Green I dye, and also by the ladder-like pattern on agarose-gel electrophoresis. Key results The assay was found to be highly specific for DNA templates of pig origin and showed no cross-reactivity with other food animals, viz. cattle, buffalo, sheep, and goats. The analytical sensitivities of the LAMP and PCR assays were recorded as up to 0.00001 ng and 0.1 ng respectively, of the absolute DNA content. The laboratory validation of the developed method was performed on blind samples and an admixture of meat from different food animals, viz. cattle, buffalo, sheep, goat and pig. The analysis could be performed in an hour by using supernatant from Phire Animal Tissue Direct PCR kit-treated tissue, excluding the complex process of nucleic acid extraction. Conclusion The LAMP assay was found to be cost-effective, easy to perform, and highly species-specific for pig tissue in meat and meat admixture. The result of the assay can be analysed with the naked eye without the need for sophisticated equipment. Compared with pre-standardised PCR assay, the developed LAMP method was quite sensitive and could be performed within 1 h, from sampling to analysis results. Implications The developed LAMP assay is low resource-based single-tube approach that could be exploited significantly in the fields of diagnostics, agriculture, and aquaculture.

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Specific and Sensitive Detection of Mycocentrospora acerina (Hart.) Causing Round Leaf Spot Disease in Sanqi (Panax notoginseng)

Round leaf spot, caused by Mycocentrospora acerina, is one of the most destructive diseases in Sanqi (Panax notoginseng) plantations in China. Accurate and timely detection of M. acerina is critical for developing effective integrated disease management strategies. Therefore, we developed a loop-mediated isothermal amplification (LAMP) assay for detection of M. acerina with primers targeting the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA). The LAMP reaction products were visually assessed using SYBR Green I and agarose gel electrophoresis. The ideal reaction temperature and time of LAMP assay were optimized to 64.5 °C and 45 min, respectively. The specificity of the developed LAMP assay was validated using 78 isolates belonging to 26 species, including M. acerina, Mycocentrospora species, and other plant pathogens. The LAMP assay was highly specific for M. acerina. Positive reactions were obtained only with the genomic DNA of M. acerina, and no cross-reaction was obtained with DNA extracted from other species. The detection limit of the LAMP assay for M. acerina was 10 fg genomic DNA per 25-μL reaction mixture. The LAMP assay successfully detected M. acerina in both symptomatic and latently infected leaf samples. The results indicate that the LAMP assay has the potential to be an efficient, highly specific, and sensitive method for diagnosing P. notoginseng round leaf spot disease caused by M. acerina in both the symptomatic and latent stages in the field and might be useful for disease management.

Loop-mediated isothermal amplification for detecting the Ile-2041-Asn mutation in fenoxaprop-P-ethyl-resistant Alopecurus aequalis.

Shortawn foxtail (Alopecurus aequalis Sobol.), a competitive grass weed severely infesting overwintering crops worldwide, has evolved resistance to the highly efficient acetyl-CoA carboxylase (ACCase)-inhibiting herbicide fenoxaprop-P-ethyl. The Ile-to-Asn substitution at codon position 2041 of ACCase is a dominant resistance mutation that has been associated with fenoxaprop-P-ethyl resistance in A.aequalis. However, its detection based on conventional methods such as polymerase chain reaction (PCR) and gene sequencing is rather labor- and time-consuming. In order to facilitate its detection in field populations of A.aequalis, a simple and efficient method with high sensitivity to the Ile-2041-Asn mutation was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed to target a 244-bp fragment of ACCase comprising codon position 2041. Using the special primers and genomic DNA of A.aequalis, the concentrations of reaction components, temperature and time each were optimized. The LAMP reaction for the detection of the Ile-2041-Asn mutation was processed at 65 °C for 45 min followed by 80 °C for 10min to stop the reaction. The LAMP method developed was 1000-fold more sensitive than the conventional PCR method, and the detection was also practicable when using crude DNA of A.aequalis as a template. The low cost, simplicity and high sensitivity of the developed LAMP assay make the detection of the Ile-2041-Asn mutation easier and quicker, which may contribute to the monitoring and management of resistance development to fenoxaprop-P-ethyl in A.aequalis. © 2022 Society of Chemical Industry.

Molecular detection of “Candidatus Rickettsia tarasevichiae” by Loop-mediated Isothermal Amplification (LAMP) of the ompA gene

“Candidatus Rickettsia tarasevichiae” (CRT) is increasingly being recognized as a disease causative agent in China and poses a great challenge to public health. Rapid and accurate detection is indispensable for laboratory diagnosis of infection caused by CRT and its surveillance in ticks. In the present study, a novel DNA-based loop-mediated isothermal amplification (LAMP) assay targeting the ompA gene was developed for the detection of CRT in tick samples. A set of universal primers specific to CRT were designed using PrimerExplorer V5 software. The analytical sensitivity, evaluated using recombinant plasmids containing the ompA gene, reached up to 1 copy per reaction, greater than that of the PCR assay targeting the same gene. This LAMP assay specifically detected CRT and showed no cross-reaction with other species common in China within the genus Rickettsia. In addition, this newly developed LAMP assay presented high diagnostic sensitivity of CRT detection validated by known positive DNA samples from ticks and simulated clinical samples. The applicability of the LAMP assay was evaluated by screening CRT from ticks, and the result showed that CRT circulation in Weichang County, China, was confirmed. Our findings indicate that this LAMP method is sensitive and specific for the detection of CRT and may have a potential application in the detection of CRT infection in patients and ticks.

Rapid and Visual Detection of Toxoplasma gondii in Blood Samples from Pet Cats and Dogs by Loop-Mediated Isothermal Amplification.

Background: Toxoplasma gondii is an obligate intracellular parasite that invades nearly all nucleated cells of a broad spectrum of vertebrate hosts, and which may cause serious disease in immunocompromised patients, as well as in the immunologically incompetent fetus. This study aimed to establish a loop-mediated isothermal amplification (LAMP) technique to rapidly detect T. gondii in the blood infection by targeting the 529 bp repeat element of T. gondii. Materials and Methods: A turbidity monitoring system, together with visual reagent, was used to test the amplification result of the LAMP assay. In addition, the specificity and sensitivity of the LAMP assay were measured. Results: The results suggest that the successfully established LAMP assay profile can detect the DNA of T. gondii at 67°C within 40 min. The limit of detection of the LAMP assay was 101 copies/μL. No cross reaction occurred with Plasmodium vivax, Toxocara cati, Clonorchis sinensi, Spirometra mansoni or Cryptosporidium parvum. We validated the developed LAMP assay by detecting T. gondii in DNA extracted from 353 blood samples collected from domestic cats and dogs. The percentages of positive results in detecting these blood samples by LAMP and conventional PCR were 5.38% and 2.83%, respectively. Conclusions: Our findings show that the developed LAMP assay offers higher analytical sensitivity than conventional PCR and good analytical specificity, minimizes aerosol contamination, and can be applied to on-site rapid detection of T. gondii.

Rapid and visual detection of soybean mosaic virus SC7 with a loop-mediated isothermal amplification strategy

Soybean mosaic virus (SMV) is the most prevalent viral pathogen of soybean worldwide that reduces seed quality and causes serious losses in yields. The accurate, rapid and early detection of SMV strains is crucial for screening SMV-resistant soybean varieties and controlling soybean mosaic disease timely. In this study, we report a loop-mediated isothermal amplification (LAMP) assay for visual and rapid detection of SMV-SC7, the major prevalent SMV strain in China, in soybean leaves. Specific primers were rationally designed and tested to detect the SMV-SC7 strain. The detection limit for SMV-SC7 can be as low as 10−4 ng/μL by the developed LAMP assay. These results were visualized by three indicators (SYBR green I, neutral red and hydroxy naphthol blue) with high fidelity. The high analytical performance of the LAMP assay was demonstrated with 21 soybean cultivars, among which 8 soybean cultivars were further inoculated with consistent results to those achieved by LAMP. This visual and rapid LAMP assay is highly promising for early detection of SMV-SC7-associated plant diseases.

Detection of Campylobacter jejuni Based on a Real-Time Fluorescence Loop-Mediated Isothermal Amplification Method.

Campylobacter jejuni (C. jejuni), a foodborne pathogenic bacterium, is among the most prevalent causes of human gastroenteritis globally. We developed and evaluated a loop-mediated isothermal amplification (LAMP) method to detect C. jejuni. Outer primers and inner primers were designed based on the hipO gene. The ratio between the concentrations of the inner and outer primers and the reaction temperature were then optimized to achieve optimal assay conditions. The analytical specificity tests showed that, among 12 genera of 74 pure bacterial culture strains, only four C. jejuni isolates could be detected, whereas no amplification was observed in C. coli, C. lari, and the other 11 genera of foodborne pathogens (n = 70). Moreover, the LAMP assay showed a higher analytical sensitivity (34.2 fg μL−1) than the conventional PCR method (342 fg μL−1). The limit of detection of C. jejuni based on the LAMP assay was 103 CFU g−1 in the artificially spiked samples of chicken meat. In conclusion, the developed LAMP assay will be a powerful and practical tool for the fast, specific, and sensitive detection of C. jejuni.

Loop-Mediated Isothermal Amplification for the Rapid Detection of the Mutation of Carbendazim-Resistant Isolates in Didymella bryoniae

Gummy stem blight (GSB) caused by Didymella bryoniae (D. bryoniae) is a worldwide fungal soil-borne disease that can cause severe yield reduction of watermelon. To shorten the monitoring time of carbendazim-resistant strains of D. bryoniae in the field, in this study, we developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection of carbendazim-resistant strains of D. bryoniae. The β-tubulin gene of carbendazim-resistant strains was selected as the target for primer design. Based on the color change of hydroxy naphthol blue (HNB) and gel electrophoresis, the optimal reaction conditions for LAMP were determined at 65 °C for 50 min. In specificity tests, the LAMP assay was able to distinguish between carbendazim-resistant and sensitive strains of D. bryoniae. Moreover, in sensitivity tests, the detection limit was 1 ng/μL D. bryoniae DNA of the carbendazim-resistant strain. In addition, the LAMP method was successfully applied to detect carbendazim-resistant strains in D. bryoniae-infested samples. Therefore, the developed LAMP assay provides a new method for the rapid detection of carbendazim-resistant strains of D. bryoniae.

Loop-mediated isothermal amplification (LAMP) assays for detection of the New Guinea fruit fly Bactroceratrivialis (Drew) (Diptera: Tephritidae)

The cue-lure-responding New Guinea fruit fly, Bactroceratrivialis, poses a biosecurity risk to neighbouring countries, e.g., Australia. In trapping programs, lure caught flies are usually morphologically discriminated from non-target species; however, DNA barcoding can be used to confirm similar species where morphology is inconclusive, e.g., Bactrocerabreviaculeus and B.rufofuscula. This can take days—and a laboratory—to resolve. A quicker, simpler, molecular diagnostic assay would facilitate a more rapid detection and potential incursion response. We developed LAMP assays targeting cytochrome c oxidase subunit I (COI) and Eukaryotic Translation Initiation Factor 3 Subunit L (EIF3L); both assays detected B.trivialis within 25 min. The BtrivCOI and BtrivEIF3L assay anneal derivatives were 82.7 ± 0.8 °C and 83.3 ± 1.3 °C, respectively, detecting down to 1 × 101 copies/µL and 1 × 103 copies/µL, respectively. Each assay amplified some non-targets from our test panel; however notably, BtrivCOI eliminated all morphologically similar non-targets, and combined, the assays eliminated all non-targets. Double-stranded DNA gBlocks were developed as positive controls; anneal derivatives for the COI and EIF3L gBlocks were 84.1 ± 0.7 °C and 85.8 ± 0.2 °C, respectively. We recommend the BtrivCOI assay for confirmation of suspect cue-lure-trapped B.trivialis, with BtrivEIF3L used for secondary confirmation when required.

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid visual detection of porcine circovirus type 2 (PCV2) and its application.

A cost effective, simple and rapid method is critical for detection of porcine circovirus type 2 (PCV2) infection in pigs. The present study reports the development and evaluation a loop-mediated isothermal amplification (LAMP) assay for rapid visual detection of PCV2 in pigs. The time and temperature conditions for amplification of PCV2 genes were optimized to be 30 min at 67 °C. The developed assay was 10 fold more sensitive than conventional PCR with analytical sensitivity of 5 pg and 50 pg, respectively. The developed LAMP assay had a sensitivity of 100%, specificity of 85.45% and overall accuracy of 89.70%. This is perhaps the most rapid of all LAMP reports for PCV2 detection available globally. The assay did not cross-react with porcine parvovirus or classical swine fever virus. DNA sequencing was done to ensure accuracy of LAMP assay results. The assay was assembled into a kit of 20 reactions and validated in different laboratories in India. The developed LAMP assay was proved to be a specific, sensitive and rapid method for visual detection of PCV2 which does not require costly equipments.

Development and application of a loop-mediated isothermal amplification method for rapid detection of Pasteurella multocida

Pasteurella multocida is an important pathogen affecting livestock and poultry causing significant economic losses. A loop-mediated isothermal amplification (LAMP) assay using four primers targeting a conserved region of the kmt1 gene was standardised to diagnose Pasteurella multocida. The test was carried out at 64oC for 45 minutes, the LAMP products could be visually confirmed using fluorescent dye SYBR Green I as detection reagent both with naked eye as well as under UV-illumination. The sensitivity of the developed LAMP assay was 104 fold higher than PCR. Furthermore, no cross-reactivity was found with the other tested bacteria. The developed LAMP assay allows easy, rapid, accurate and sensitive detection of Pasteurella multocida.

Optimization for Rapid Detection of E. coli O157:H7 Using Real-Time Loop-Mediated Isothermal Amplification

Background: E. coli O157:H7-related food poisoning is one of the most well-known causes of bloody diarrhea illness around the world. Therefore, devising a rapid, highly sensitive, and convenient detection technique for this species is crucial. In this work, we optimized a colorimetric Loop-mediated isothermal amplification (LAMP) for detecting the intimin gene from E. coli O157:H7. Methods: In this study, eae (intimin), one of the virulence factors of E. coli O157:H7, was selected as the target gene, and specific primers were designed for the sequence of this gene using the Primer Explorer V5 software. The LAMP reaction was optimized with three variable factors of MgSO4 concentration, temperature, and incubation time, in a traditional (separate) way and by Taguchi experimental design. Finally, the LAMP products were visualized by 2% agarose gel electrophoresis stained with ethidium bromide or green fluorescence (SYBR green I) and the pink fluorescence (KBC power load), which can be observed using the naked eye. Results: The LAMP reaction was optimized at 63°C and 8 mM MgSO4 for 40 min. Also, the naked eye can readily visualize the LAMP products within 40 minutes and have a detection limit of 3.2×104 CFU/mL according to 0.38 fg from the genome. Designed primers based on the gene sequence proved their specificity by testing 4 species of other common foodborne pathogenic microorganisms. Conclusion: The rapid, sensitive, one-step-visually developed LAMP assay could be of interest for screening functions in food analytical laboratories without special equipment or trained personnel.

A versatile isothermal amplification assay for the detection of leptospires from various sample types.

BackgroundLeptospirosis is a zoonotic disease caused by bacteria of the genus Leptospira that affects both humans and animals worldwide. Early detection of the pathogen in humans is crucial for early intervention and control of the progression of the disease to a severe state. It is also vitally important to be able to detect the presence of the pathogen in carrier animals to control the spread of the disease from the environment. Here we developed a simple and rapid loop-mediated isothermal amplification (LAMP) assay targeting the leptospiral secY gene.ResultsSeveral reaction conditions of the LAMP reaction were optimized to ensure efficient amplification of the target DNA. The sensitivity of the developed LAMP assay obtained using a pure Leptospira culture was 2 × 104 copies of genomic DNA per reaction (equivalent to 0.1 ng) for a 40-minute reaction time. No cross-reactions were observed in the LAMP reaction against a series of non-leptospiral bacteria, indicating a specific reaction. The applicability of the LAMP assay was demonstrated on human blood and urine specimens collected from suspected leptospirosis patients and rat kidney specimens collected from suspected leptospirosis outbreak areas and high-risk areas. The developed LAMP assay demonstrated a higher detection rate for leptospiral DNA compared with the polymerase chain reaction (PCR) assay, possibly due to the presence of inhibitory substances, especially in rat kidney specimens, to which the PCR method is more susceptible. The present findings also highlight the importance of urine sample collection from patients for routine monitoring of the disease.ConclusionsIn short, the developed LAMP assay can serve as a feasible alternative tool for the diagnosis of leptospirosis and be used for epidemiological and environmental surveillance of the disease, considering its robustness, rapidity, sensitivity, and specificity, as demonstrated in this study.