Loop-mediated Isothermal Amplification Method Research Articles

Development and Validation of a Loop-Mediated Isothermal Amplification (LAMP) Method for the Rapid, Sensitive, and Specific Detection of Hazelnut as an Allergen in Food Matrix.

Analytical verification of hazelnut in food supports risk-based approaches for proper allergen labeling to prevent unwanted allergic reactions or to quality control diagnostic or therapeutic allergen preparations for allergic subjects. We present the development and validation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of hazelnut by targeting the internal transcribed spacer 2 gene. The qualitative method requires neither sophisticated analytical equipment nor antibodies, allowing an easy-to-use application with no ethical concern related to the use of animals. It demonstrated a limit of detection at or below 10 mg/kg hazelnut in various food matrices, making it also suitable for verifying hazelnut at levels of clinically relevant eliciting doses. Validation against proficiency test samples and testing of applicability with commercial food items confirmed its usefulness in processed foods. The simplicity of the method, including visual colorimetric detection, combined with high specificity and sensitivity, represents an advancement over existing qualitative methods.

Optimization of loop-mediated isothermal amplification assay for sunflower mildew disease detection

Loop-Mediated Isothermal Amplification (LAMP) represents a valuable technique for DNA/RNA detection, known for its exceptional sensitivity, specificity, speed, accuracy, and affordability. This study focused on optimizing a LAMP-based method to detect early signs of Plasmopara halstedii, the casual pathogen of sunflower downy mildew, a severe threat to sunflower crops. Specifically, a set of six LAMP primers (two outer, two inner, and two loop) were designed from P. halstedii genomic DNA, targeting the ribosomal Large Subunit (LSU). These primers were verified by in silico analysis and experimental validation using both target and non-target species' DNAs. Optimizations encompassing reaction conditions (temperature, time) and component concentrations (magnesium, Bst DNA polymerase, primers, and dNTP) were determined. Validation of these optimizations was performed by agarose gel electrophoresis. Furthermore, various colorimetric chemicals (Neutral Red, Hydroxynaphthol Blue, SYBR Safe, Thiazole Green) were evaluated to facilitate method analysis, and the real-time analysis has been optimized, presenting multiple approaches for detecting sunflower downy mildew using the LAMP technique. The analytical sensitivity of the method was confirmed by detecting P. halstedii DNA concentrations as low as 0.5 pg/μl. This pioneering study, establishing P. halstedii detection through the LAMP method, stands as unique in its field. The precision, robustness, and practicality of the LAMP protocol make it an ideal choice for studies focusing on sunflower mildew, emphasizing its recommended use due to its operational ease and reliability.

A colorimetric reverse-transcription loop-mediated isothermal amplification method targeting the L452R mutation to detect the Delta variant of SARS-CoV-2

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered global difficulties for both individuals and economies, with new variants continuing to emerge. The Delta variant of SARS-CoV-2 remains most prevalent worldwide, and it affects the efficacy of coronavirus disease 2019 (COVID-19) vaccination. Expedited testing to detect the Delta variant of SARS-CoV-2 and monitor viral transmission is necessary. This study aimed to develop and evaluate a colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) technique targeting the L452R mutation in the S gene for the specific detection of the Delta variant. In the test, positivity was indicated as a color change from purple to yellow. The assay’s 95% limit of detection was 57 copies per reaction for the L452R (U1355G)-specific standard plasmid. Using 126 clinical samples, our assay displayed 100% specificity, 97.06% sensitivity, and 98.41% accuracy in identifying the Delta variant of SARS-CoV-2 compared to real-time RT-PCR. To our knowledge, this is the first colorimetric RT-LAMP assay that can differentiate the Delta variant from its generic SARS-CoV-2, enabling it as an approach for studying COVID-19 demography and facilitating proper effective control measure establishment to fight against the reemerging variants of SARS-CoV-2 in the future.

Development of machine learning algorithm for loop-mediated isothermal amplification including influence of temperature

To predict amplification in the Loop-Mediated Isothermal Amplification (LAMP) method, a binary model has been developed. This model assesses whether the combination of partial template binding facilitated by multiple primers aligns with a specific signature indicating amplification. For this model, 1512 settings were adjusted, encompassing variations in the extraction oligomer position from the primer, the oligomer length, the number of allowed mismatched bases, and the stringency of the homology position. The optimal set was determined based on the F1 value at a reaction temperature of 60°C, resulting in a value of 0.58. To implement machine learning by amalgamating multiple models, duplicate models were eliminated through cluster analysis. Machine learning using AutoML was performed on 108 distinct models. Consequently, the F1 values were 0.7119 at a reaction temperature of 60°C and 0.7210 at 68°C. The weak correlation between feature importance and the F1 value of the model alone suggests that machine learning utilizing this model incorporates numerous models to enhance prediction accuracy.

Development of a real-time loop-mediated isothermal amplification method with toothpick sampling for non-destructive detection of Ustilago esculenta in Zizania latifolia

Development of a real-time loop-mediated isothermal amplification method with toothpick sampling for non-destructive detection of Ustilago esculenta in Zizania latifolia

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.

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.

A colorimetric assay for Neospora caninum utilizing the loop-mediated isothermal amplification technique

Neospora caninum (N. caninum) is a protozoan parasite that poses a serious risk to livestock by infecting various domestic and wild animals. Loop-Mediated Isothermal Amplification (LAMP) offers a cost-effective, highly sensitive, and specific method for detecting protozoan parasites. This study aims to develop a precise, rapid, and visually assessable colorimetric LAMP method, improving on traditional techniques. We employed a rigorous screening process to identify the optimal primer set for this experiment. Subsequently, we fine-tuned the LAMP reaction at 65 °C for 40 min with 270 μmol/L neutral red. We then confirmed the specificity of primers for N. caninum through experimental validation. The LAMP method demonstrated a lower detection limit compared to traditional Polymerase Chain Reaction (PCR) techniques. While LAMP offers clear advantages, the prevalence of DNA detected in 89 sheep serum and 59 bovine serum samples using the nested PCR method was 3.37 % (3/89) and 1.69 % (1/59), respectively. In contrast, when the LAMP method was employed, the prevalence of detected DNA rose to 5.61 % (5/89) for sheep and 3.38 % (2 /59) for bovine. A comparison of two molecular assays using the intragroup correlation coefficient (ICC) resulted in a value of 0.999 (95 % CI: 0.993–0.996, p < 0.001), indicating the LAMP method is in the “better” range according to James Lee's categorization. The LAMP technique, optimized with specific primers of N. caninum and neutral red dye, not only exhibited higher sensitivity but also provided convenience over conventional PCR methods, highlighting its potential for on-site applications and cost-effective field detection.

Identification of Pathogen Causing Bulb Rot in Fritillaria taipaiensis P. Y. Li and Establishment of Detection Methods.

Fritillaria taipaiensis P. Y. Li (F. taipaiensis) is a traditional Chinese herbal medicine that has been used for over two millennia to treat cough and expectoration. However, the increasing cultivation of F. taipaiensis has led to the spread of bulb rot diseases. In this study, pathogens were isolated from rotten F. taipaiensis bulbs. Through molecular identification, pathogenicity testing, morphological assessment, and microscopy, Fusarium solani was identified as the pathogen causing bulb rot in F. taipaiensis. The colonization of F. solani in the bulbs was investigated through microscopic observation. The rapid and accurate detection of this pathogen will contribute to better disease monitoring and control. Loop-mediated isothermal amplification (LAMP) and qPCR methods were established to quickly and specifically identify this pathogen. These results provide valuable insights for further research on the prediction, rapid detection, and effective prevention and control of bulb rot in F. taipaiensis.

Molecular Detection of Respiratory Tract Viruses in Chickens at the Point of Need by Loop-Mediated Isothermal Amplification (LAMP).

Accurate and timely molecular diagnosis of respiratory diseases in chickens is essential for implementing effective control measures, preventing the spread of diseases within poultry flocks, minimizing economic loss, and guarding food security. Traditional molecular diagnostic methods like polymerase chain reaction (PCR) require expensive equipment and trained personnel, limiting their use to centralized labs with a significant delay between sample collection and results. Loop-mediated isothermal amplification (LAMP) of nucleic acids offers an attractive alternative for detecting respiratory viruses in broiler chickens with sensitivity comparable to that of PCR. LAMP's main advantages over PCR are its constant incubation temperature (∼65 °C), high amplification efficiency, and contaminant tolerance, which reduce equipment complexity, cost, and power consumption and enable instrument-free tests. This review highlights effective LAMP methods and variants that have been developed for detecting respiratory viruses in chickens at the point of need.

Rapid cyprinid herpesvirus 3 detection using loop-mediated isothermal amplification (LAMP) combined with gold nanoprobes and SYBR safe

As an etiological agent of a notifiable and emerging disease, Cyprinid herpesvirus 3 (CyHV-3 or koi herpesvirus, KHV) is a highly risky factor that affects koi and common carp yield through increasing the mortality rates. In the current study, a highly rapid and sensitive technique was established to detect CyHV-3 with the help of loop-mediated isothermal amplification (LAMP) method with dual visualizing indicators (SYBR safe and gold nanoparticle probes; AuNP-probes). Six specific primers were used to amplify the thymidine kinase (TK) of CyHV-3 using a LAMP reaction of 45 min at 65 °C along with detecting the products through visual inspection via hybridization at 65 °C for 20 min with a functionalized thiol-AuNP probe. A closed-tube LAMP-SYBR safe assay was also developed under the same condition without AuNP probe. The amplification of KHV-LAMP products were evaluated by the naked eye, through fluorescent emission of LAMP-SYBR safe dye-complex and colorimetric aggregate of LAMP product in the presence of AuNP probe. Furthermore, quantitative measurement was applied to the LAMP-AuNPs assay, using the spectral shift assay. The detection limit was 1.25 fentograms DNA which can compete the latest techniques available. The proposed technique yielded negative outcomes with DNA template from other viruses such as CyHV-1 and CEV. The LAMP-SYBR safe and LAMP-AuNPs assays introduced in this article were very sensitive, fast, and reliable diagnostic tools for the detection of CyHV-3.

Multiplex LAMP assay for detecting the prevalent species of dust mites Dermatophagoides farinae and Dermatophagoides pteronyssinus in the domestic environment

Dermatophagoides farina (D. farinae) and Dermatophagoides pteronyssinus (D. pteronyssinus) are the prevalent kinds of house dust mites (HDMs). HDMs are common inhalant allergens that cause a range of allergic diseases, such as rhinitis, atopic dermatitis, and asthma. The epidemiology of these diseases is associated with exposure to mites. Therefore, in the present study, a method named multiplex loop-mediated isothermal amplification (LAMP) was developed to detect environmental dust mites. The multiplex LAMP assay allows amplification within a single tube and has an ITS plasmid detection limit as low as 40 fg/µL for both single dust mites and mixed dust mites (D. pteronyssinus and D. farinae), which is up to ten times more sensitive than classical PCR techniques. Furthermore, the multiplex LAMP method was applied to samples of single dust mites and clinical dust to confirm its validity. The multiplex LAMP assay exhibited higher sensitivity, simpler instrumentation, and visualization of test results, indicating that this method could be used as an alternative to traditional techniques for the detection of HDMs.

Development of a LAMP method with lateral flow DNA chromatography to diagnose toxoplasmosis in immunocompromised patients

BackgroundRapid and accurate diagnosis of toxoplasmosis is critical, particularly for immunocompromised patients. Several molecular methods could have value for toxoplasmosis diagnosis, but often require sophisticated and expensive equipment, and as such are impractical for use in resource-limited countries. Our study aimed to develop a new rapid diagnostic test for toxoplasmosis that can be used in developed countries as well as low- or middle-income countries.MethodsCommon primers for conventional loop-mediated isothermal amplification (LAMP) and the new LAMP DNA chromatography method were designed based on a 529-bp repeat present in Toxoplasma gondii genomic DNA. A total of 91 clinical samples from 44 patients suspected of having toxoplasmosis who were treated at several hospitals across Japan were tested using the new LAMP DNA chromatography method, conventional LAMP, and nested PCR and the sensitivity and specificity of the methods was compared.ResultsThe LAMP DNA chromatography method showed better sensitivity and specificity (68.2% and 100%, respectively) compared with the nested PCR (45.4% and 100%, respectively) and conventional LAMP (63.6% and 100%, respectively) methods for diagnosis of toxoplasmosis in immunocompromised patients. LAMP DNA chromatography also has better sensitivity and specificity (75% and 100%, respectively) than nested PCR (50.0% and 93.5%, respectively) and conventional LAMP (62.5% and 100%, respectively) to diagnose toxoplasma encephalitis using CSF samples.ConclusionWe developed a LAMP DNA chromatography method to detect T. gondii DNA in clinical samples. This method also successfully detected T. gondii DNA in CSF from patients with toxoplasma encephalitis. This newly developed method can be a valuable rapid diagnostic test for toxoplasmosis in a range of settings, including resource-limited areas like those in low- or middle-income countries.

A Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Didymella segeticola Causing Tea Leaf Spot.

Tea leaf spot caused by Didymella segeticola is an important disease that threatens the healthy growth of tea plants (Camellia sinensis) and results in reductions in the productivity and quality of tea leaves. Early diagnosis of the disease is particularly important for managing the infection. Loop-mediated isothermal amplification (LAMP) assay is an efficient diagnostic technique with the advantages of simplicity, specificity, and sensitivity. In this study, we developed a rapid, visual, and high-sensitivity LAMP assay for D. segeticola detection based on sequence-characterized amplified regions. Two pairs of amplification primers (external primers F3 and B3 and internal primers FIP and BIP) were designed based on a specific sequence in D. segeticola (NCBI accession number: OR987684). Compared to common pathogens of other genera in tea plants and other species in the Didymella genus (Didymella coffeae-arabicae, Didymella pomorum, and Didymella sinensis), the LAMP method is specific for detecting the species D. segeticola. The assay was able to detect D. segeticola at a minimal concentration of 1 fg/μL genomic DNA at an optimal reaction temperature of 65 °C for 60 min. When healthy leaves were inoculated with D. segeticola in the laboratory, the LAMP method successfully detected D. segeticola in diseased tea leaves at 72 h post inoculation. The LAMP assays were negative when the DNA samples were extracted from healthy leaves. Leaf tissues with necrotic lesions from 18 germplasms of tea plants tested positive for the pathogen by the LAMP assay. In summary, this study established a specific, sensitive, and simple LAMP method to detect D. segeticola, which provides reliable technical support for estimating disease prevalence and facilitates sustainable management of tea leaf spot.

Molecular detection of toxoplasmosis in wild rats using loop-mediated isothermal amplification assay.

Toxoplasmosis is caused by the parasite Toxoplasma gondii. Cats are the only known hosts that excrete resistant oocysts. Wild rats serve as crucial reservoirs and intermediate hosts for T. gondii's survival and dissemination. Consuming soil and water containing oocysts can lead to illness. This study aimed to estimate the prevalence of toxoplasmosis in wild rats through molecular detection as an indicator of environmental contamination in Surabaya. One hundred rats were collected from the three areas (housing, dense settlements, and traditional markets) and distributed into the five zones: West, East, Central, North, and South of Surabaya. Brain tissue samples were extracted using a Geneaid™ (New Taipei City, Taiwan) DNA isolation kit and analyzed through the loop-mediated isothermal amplification (LAMP) method. The study analyzed brain tissue from 100 wild rats, consisting of 77 Rattus tanezumi and 33 Rattus norvegicus, displaying 30% LAMP positivity. The study revealed that 30% (30/100) of wild rats tested were infected with T. gondii. The molecular prevalence rate in male rats was 32.35% (22/68), compared to females with 25% (8/32). 41.9% of the housing population, 33.3% of traditional markets, and 22.6% of dense settlements had the highest molecular prevalence. The high positive molecular rate at the trapping site can be attributed to cats and dense populations. Thirty percentage wild rats were tested positive for toxoplasmosis in Surabaya, East Java, Indonesia using LAMP method. Implementing strict control and monitoring is crucial in preventing the transmission of diseases from wild rats to humans. It is necessary to carry out further research related to genetic analysis of T. gondii to determine the type of T. gondii that infects animals and humans in Surabaya through bioassay and molecular test.

Direct detection of Aeromonas hydrophila from water and tissue samples by loop-mediated isothermal amplification (LAMP) method

Direct detection of Aeromonas hydrophila from water and tissue samples by loop-mediated isothermal amplification (LAMP) method

Development and Comparison of Visual LAMP and LAMP-TaqMan Assays for Colletotrichum siamense.

Strawberry anthracnose caused by Colletotrichum spp. has resulted in significant losses in strawberry production worldwide. Strawberry anthracnose occurs mainly at the seedling and early planting stages, and Colletotrichum siamense is the main pathogen in North China, where mycelia, anamorphic nuclei, and conidia produced in the soil are the main sources of infection. The detection of pathogens in soil is crucial for predicting the prevalence of anthracnose. In this study, a visualized loop-mediated isothermal amplification (LAMP) assay and a loop-mediated isothermal amplification method combined with a TaqMan probe (LAMP-TaqMan) assay were developed for the β-tubulin sequence of C. siamense. Both methods can detect Colletotrichum siamense genomic DNA at very low concentrations (104 copies/g) in soil, while both the visualized LAMP and LAMP-TaqMan assays exhibited a detection limit of 50 copies/μL, surpassing the sensitivity of conventional PCR and qPCR techniques, and both methods showed high specificity for C. siamense. The two methods were compared: LAMP-TaqMan exhibited enhanced specificity due to the incorporation of fluorescent molecular beacons, while visualized LAMP solely necessitated uncomplicated incubation at a constant temperature, with the results determined by the color change; therefore, the requirements for the instrument are relatively straightforward and user-friendly. In conclusion, both assays will help monitor populations of C. siamense in China and control strawberry anthracnose in the field.

Rapid and quantitative detection of Aspergillus niger Van Tieghem using loop-mediated isothermal amplification assay

AbstractPeanut (Arachis hypogaea L.) crown rot and root rot are common diseases caused by Aspergillus niger Van Tieghem. Early and accurate detection of A. niger is key to disease management. In this study, the design of two to five sets of loop-mediated isothermal amplification (LAMP) primers was based on the EglA, GOD, Tub, NRPS, Tan, CbhA, and CbhB genes of A. niger. Of these, primer set GOD-91 was selected for optimization of the three-factor LAMP system: the Bst DNA polymerase concentration, the concentration ratio of the inner and outer primers, and the concentration of Mg2+. In addition, the optimized LAMP reaction system for A. niger detection was validated for specificity, sensitivity, and on-site feasibility. The specificity test showed that A. niger could be specifically detected with the proposed method without cross-amplification of other pathogenic fungi DNA. Moreover, based on the sensitivity test, the lowest detection limit of this reaction system was 5.1 × 10−7 ng/µL pAN01 plasmid DNA, after which a standard curve was generated for the quantitative detection of A. niger. The LAMP method was further applied for field sample assessment before and after A. niger infection, successfully detecting A. niger presence in the samples collected in the field. This study yielded a sensitive, specific, and reproducible LAMP system that can be used to assess on-site samples within 45 min. It is an effective approach for the rapid and quantitative detection of A. niger.

A phosphorylated guanidine chitosan and UiO-66-NH2 modified magnetic nanoparticle platform for enrichment and detection of multiple bacteria

Wastewater-based epidemiology (WBE) is a powerful tool for early warning of infectious disease outbreaks. Hence, a rapid and portable pathogen monitoring system is urgent needed for on-site detection. In this work, we first reported synthesis of an artificial modulated wide-spectrum bacteria capture nanoparticle (Arg-CSP@UiO@Fe3O4). Arginine-modified phosphorylated chitosan (Arg-CSP) coating could provide strongly positive charged guanidinium group for pathogen interaction by electrostatic attraction, and UiO-66-NH2 layer could help Arg-CSP graft onto Fe3O4 magnetic particles. The capture efficiency of Arg-CSP@UiO@Fe3O4 reached 92.2 % and 97.3 % for Escherichia coli (E.coli) and Staphylococcus epidermidis (S.epidermidis)within 40 min, in 10 mL sample. To prevent pathogen degradation in sewage, a portable nucleic acid extraction-free method was also developed. UiO-66-NH2 could disintegrate in buffer with high concentration of PO43− for bacterium desorption, and then nucleic acid of the bacteria was released by heating. The DNA template concentration obtained by this method was 779.28 times higher than that of the direct thermal lysis product and 8.63 times higher than that of the commercial kit. Afterwards, multiple detection of bacteria was realized by loop-mediated isothermal amplification (LAMP). Artificial regulated pathogen desorption could prevent non-specific adsorption of nucleic acid by nanoparticles. The detection limit of Arg-CSP@UiO@Fe3O4-LAMP method was 80 cfu/mL for E.coli and 300 cfu/mL for S.epidermidis. The accuracy and reliability of the method was validated by spiked sewage samples. In conclusion, this bio-monitoring system was able to detect multiple bacteria in environment conveniently and have good potential to become an alternative solution for rapid on-site pathogen detection.

The Establishment of the Multi-Visual Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Vibrio harveyi, Vibrio parahaemolyticus, and Singapore grouper iridovirus

Groupers are valuable economic fish in the southern sea area of China, but the threat of disease is becoming more and more serious. Vibrio harveyi, V. parahaemolyticus, and Singapore grouper iridovirus (SGIV) are three important pathogens that cause disease in groupers, and infection with either a single one or a mix of these pathogens poses a serious threat to the healthy development of grouper culture. To enhance the rapid diagnosis and screening in the early stages, it is necessary to develop rapid detection methods of these pathogens. To simultaneously and rapidly detect the three pathogens, in this study, we utilized the TolC of V. harveyi, DNAJ of V. parahaemolyticus, and RAD2 of SGIV as the target genes and established a triple visual loop-mediated isothermal amplification (LAMP) method. This LAMP method showed a detection time as fast as 30 min and a high sensitivity of 100 fg/μL. Moreover, this method exhibited strong specificity and no cross-reaction with seven types of Vibrio and Staphylococcus aureus, as well as five common viruses in aquatic animals. Then, the LAMP products were enzymically cut, and three characteristic strips were used to identify the pathogen species. The results of the clinical trials demonstrated that the method could accurately and specifically detect V. harveyi, V. parahaemolyticus, and SGIV in grouper tissues. In summary, this study successfully established a triple visual LAMP rapid detection method for V. harveyi, V. parahaemolyticus, and SGIV. The method offers several advantages including simple equipment, easy operation, rapid reaction, high specificity, high sensitivity, and visual results. It is suitable for the early and rapid diagnosis of groupers infected with V. harveyi, V. parahaemolyticus, and SGIV, thereby providing useful technical support for further application in the large-scale disease surveillance of aquaculture animals.