Real-time Loop-mediated Isothermal Amplification Research Articles

A Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Bremia lactucae in the field.

A real-time loop-mediated isothermal amplification (LAMP) assay for the detection of Bremia lactucae, the causal pathogen of Lettuce Downy Mildew (LDM), was developed and validated to aid in-field detection of airborne inoculum. Assay specificity was confirmed against a range of other pathogenic oomycete and fungal spp and sensitivity of the assay for the detection of DNA extracted from sporangia was evaluated. The B. lactucae LAMP assay reliably detected DNA equivalent to 1 spore/reaction (16.7 pg DNA/reaction). Following extraction of DNA from rotorod air-samplers to which sporangial suspensions were added, the assay reliably detected 25 sporangia/rotorod. Detection of airborne inoculum of B. lactucae collected through the season from air-samplers deployed in field plots infected with B. lactucae and also in commercial lettuce fields in Scotland over two growing seasons was assessed. The method can be deployed on samples collected from commercial lettuce production to inform disease management strategies and limit the use of unecessary prophylactic pesticide applications.

Development of a real-time fluorescent reverse transcription loop-mediated isothermal amplification assay with quenching primers for rapid detection of rubella virus

Rubella virus infection during early pregnancy sometimes causes severe birth defects termed congenital rubella syndrome. Although there are safe and effective live-attenuated vaccines, rubella has only been certified as eliminated in the Americas within the six World Health Organization regions. Rubella remains an endemic disease in many regions, and outbreaks occur wherever population immunity is insufficient. There are two main methods for diagnosis of rubella: detection of anti-rubella IgM antibodies by enzyme immunoassay and detection of the viral genome by real-time RT-PCR. Both of these methods require substantial time and effort. In the present study, a rapid rubella detection assay using real-time fluorescent reverse transcription loop-mediated isothermal amplification with quenching primers was developed. The time required for the new assay was one-half that required for a real-time RT-PCR assay. The assay had 93.6% positive percent agreement and 100% negative percent agreement for clinical specimens compared with the real-time RT-PCR assay. The new assay is considered useful for diagnosis of rubella in areas where rubella is endemic.

Real-time fluorescence loop-mediated isothermal amplification assays for detection of zoonotic malaria Plasmodium parasites

BackgroundNatural human infections by Plasmodium cynomolgi and P. inui have been reported recently and gain the substantial attention from Southeast Asian countries. Zoonotic transmission of non-human malaria parasites to humans from macaque monkeys occurred through the bites of the infected mosquitoes. The objective of this study is to establish real-time fluorescence loop-mediated isothermal amplification (LAMP) assays for the detection of zoonotic malaria parasites by combining real-time fluorescent technology with the isothermal amplification technique. MethodsBy using 18S rRNA as the target gene, the primers for P. cynomolgi, P. coatneyi and P. inui were newly designed in the present study. Four novel real-time fluorescence LAMP assays were developed for the detection of P. cynomolgi, P. coatneyi, P. inui and P. knowlesi. The entire amplification process was completed in 60 minutes, with the assays performed at 65°C. By using SYTO-9 as the nucleic acid intercalating dye, the reaction was monitored via real-time fluorescence signal. ResultsThere was no observed cross-reactivity among the primers from different species. All 70 field-collected monkey samples were successfully amplified by real-time fluorescence LAMP assays. The detection limit for P. cynomolgi, P. coatneyi and P. knowlesi was 5 × 109 copies/µL. Meanwhile, the detection limit of P. inui was 5 × 1010 copies/µL. ConclusionThis is the first report of the detection of four zoonotic malaria parasites by real-time fluorescence LAMP approaches. It is an effective, rapid and simple-to-use technique. This presented platform exhibits considerable potential as an alternative detection for zoonotic malaria parasites.

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.

Evaluation of a rapid Loop Mediated Isothermal Amplification (LAMP) test for the laboratory diagnosis of sexually transmitted infections.

Sexually transmitted infections (STIs) have seen a considerable increase in the last years and given the health burden they may represent from both a personal and community perspective, they require surveillance and prevention programmes based on a timely and decentralized diagnosis. In this context, user-friendly rapid molecular tests may represent a good trade-off between diagnostic accuracy, accessibility and affordability. In this study we evaluated the diagnostic performance of a new real-time LAMP (Loop Mediated Isothermal Amplification) method for the rapid detection and differentiation of 7 major sexually transmissible pathogens by analysing real clinical samples (genital and extra-genital matrices) from individuals with suspected STIs. The assay showed good overall diagnostic performances in terms of sensitivity, specificity and concordance with a gold-standard PCR-based molecular method. This assay, not requiring specialised laboratory technicians or expensive instrumentation, but nonetheless capable of guaranteeing accurate results, is within the reach of outpatient settings, obstetrics, and gynaecology clinic, hence ensuring on-field access to early diagnosis.

Development of a quantitative colorimetric LAMP assay for fast and targeted molecular detection of the invasive lionfish Pterois miles from environmental DNA

The Mediterranean basin has faced an increased influx of invasive species since the Suez Canal expansion in 2015. The invasive lionfish species, Pterois miles, has rapidly established new populations in the Eastern Mediterranean Sea, impacting local fish biodiversity. Here, we have developed a new, fast (< 35 min) molecular approach to detect and quantify P. miles environmental DNA (eDNA) in combination with a portable device for field-based analysis. Using a species-specific real-time colorimetric loop-mediated isothermal amplification (qcLAMP) for the cytochrome oxidase subunit 1 (COI) gene, we demonstrate a high sensitivity with a limit of detection of 0.002 ng DNA per reaction, equivalent to only 50 copies of the COI gene. The assay is specific to the target in the presence of closely related and co-occurring species, and it is quantitative over five orders of magnitude. We validated the assay using aquarium water samples and further demonstrated its utility on natural eDNA samples collected from locations around the island of Crete where P. miles had been sighted. P. miles was indeed detected in three out of nine locations, two nature reserves and a closed bay. Lack of detection in the remaining locations suggests that populations are still at a low density. We also demonstrate the feasibility of P. miles eDNA qualitative detection directly from the filter used to collect eDNA-containing particles, completely omitting DNA extraction. Overall, we present a new approach for fast and targeted eDNA quantification. The developed LAMP assay together, with the quantitative real-time colorimetric detection approach, open new possibilities for monitoring invasive P. miles in the field.

Comparison of dye-based and probe-based RT-LAMP in detection of canine astrovirus.

A rapid and sensitive assay is essential for reliable surveillance and diagnosis of canine astrovirus (CaAstV). In this study, two real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays with high sensitivity, rapidity, and reliability were developed using fluorescence dye and FRET-based assimilating probes for real-time detection of CaAstV. These assays specifically amplified the ORF2 gene of CaAstV and did not amplify any sequences from canine enterovirus. The limit of detection (LOD) of both the probe-based and dye-based RT-LAMPs was 100 copies/μL. Fluorescence signals were generated within 30 min for the lowest concentration of a standard RNA sample, which was significantly faster than that achieved by real-time fluorescence quantitative PCR (qRT-PCR) assay. When clinical samples were tested, the positive and negative agreement of the dye-based RT-LAMP assay with qRT-PCR was 87.5% (14/16) and 93.55% (29/31), respectively. The positive and negative agreement of the probe-based RT-LAMP assay with qRT-PCR was 94.11% (16/17) and 96.55% (28/29), respectively. The RT-LAMP assays developed in this study showed strong potential for use as an on-site diagnostic assay for rapid, specific, and reliable detection of CaAstV in clinical samples.

Loop-mediated isothermal amplification (LAMP) assays for rapid on-site detection of Lacticaseibacillus casei, Lacticaseibacillus paracasei, and Lacticaseibacillus rhamnosus in probiotic products

We developed the colorimetric loop-mediated isothermal amplification (LAMP) and a real-time LAMP with direct DNA extraction for the on-site detection of the three closely related Lacticaseibacillus species, such as Lacticaseibacillus casei, Lacticaseibacillus paracasei, and Lacticaseibacillus rhamnosus in probiotic products. Species-specific LAMP primers were designed targeting a unique gene in each species through comparative genomic analysis. Each LAMP primer set amplified target species specifically, distinguishing between the closely related species within the Lacticaseibacillus species. Based on the designed LAMP primers, two LAMP-based assays with direct DNA extraction were compared; their limit of detection was 103 CFU/mL for the Lacticaseibacillus species. In addition, both methods were applied to 44 commercial probiotic products and obtained identical results; it took about 45 min from sample preparation to confirming the presence or absence of the Lacticaseibacillus species. Thus, these methods can be rapid and simple in-field tests to detect L. casei, L. paracasei, and L. rhamnosus in probiotic products.

Knowledge of xylem feeders (Hemiptera: Auchenorrhyncha) in Albanian olive orchards in readiness for a potential outbreak with Xylella fastidiosa invasion

Summary Since Xylella fastidiosa first appeared in southern Italy, it has become essential to manage and limit its transmission. In areas unaffected by the bacterium, such as Albania, it is necessary to assess the risk of its spread, primarily by understanding the ecology of its potential vectors. A survey of 14 olive orchards was conducted over a period of 12 months from August 2015 and July 2016 in two Albanian counties (Vlora and Tirana) where olive production is important. The aim was to investigate the presence of Auchenorrhyncha species, with particular attention to xylem feeders, and Auchenorrhyncha species belonging to the families Aphrophoridae, Cercopidae, Cicadellidae, Issidae, Flatidae were found in both counties. In Vlora, four out of 11 collected species were Aphrophoridae and Cercopidae, whereas in Tirana, three species belonging to these families were captured. In both counties, the most abundant xylem feeder species was Philaenus spumarius. In Vlora, P. spumarius was found more frequently on olive trees than in Tirana. Philaenus spumarius and Neophilaenus campestris adults were most frequent in August, September, and October 2015, while both species were almost absent from December 2015 to March 2016. Our sampling demonstrated that Vlora region has a greater number of Auchenorrhyncha species than Tirana. Furthermore, molecular tests (real-time LAMP) showed the absence of X. fastidiosa in the tested individuals. This survey constitutes an important update on the X. fastidiosa situation in Albania, showing that the country is still free of X. fastidiosa, at least in the surveyed areas.

Moving towards on-site detection of Shiga toxin-producing Escherichia coli in ready-to-eat leafy greens

Rapid identification of Shiga toxin-producing Escherichia coli, or STEC, is of utmost importance to assure the innocuousness of the foodstuffs. STEC have been implicated in outbreaks associated with different types of foods however, among them, ready-to-eat (RTE) vegetables are particularly problematic as they are consumed raw, and are rich in compounds that inhibit DNA-based detection methods such as qPCR. In the present study a novel method based on Loop-mediated isothermal amplification (LAMP) to overcome the limitations associated with current molecular methods for the detection of STEC in RTE vegetables targeting stx1 and stx2 genes. In this sense, LAMP demonstrated to be more robust against inhibitory substances in food. In this study, a comprehensive enrichment protocol was combined with four inexpensive DNA extraction protocols. The one based on silica purification enhanced the performance of the method, therefore it was selected for its implementation in the final method. Additionally, three different detection chemistries were compared, namely real-time fluorescence detection, and two end-point colorimetric strategies, one based on the addition of SYBR Green, and the other based on a commercial colorimetric master mix. After optimization, all three chemistries demonstrated suitable for the detection of STEC in spiked RTE salad samples, as it was possible to reach a LOD50 of 0.9, 1.4, and 7.0 CFU/25 g for the real-time, SYBR and CC LAMP assays respectively. All the performance parameters reached values higher than 90 %, when compared to a reference method based on multiplex qPCR. More specifically, the analytical sensitivity was 100, 90.0 and 100 % for real-time, SYBR and CC LAMP respectively, the specificity 100 % for all three assays, and accuracy 100, 96 and 100 %. Finally, a high degree of concordance was also obtained (1, 0.92 and 1 respectively). Considering the current technological advances, the method reported, using any of the three detection strategies, demonstrated suitable for their implementation in decentralized settings, with low equipment resources.

A portable all-in-one microfluidic device with real-time colorimetric LAMP for HPV16 and HPV18 DNA point-of-care testing

Screening for high-risk human papillomavirus (HPV) infection is one of the most important preventative measures for cervical cancer. However, fast, convenient, and low-cost HPV detection remains challenging, especially in resource-limited settings. Here, we report a portable all-in-one device (PAD) for point-of-care testing (POCT) for HPV16 and HPV18 DNA in cervical swabs. The PAD was engineered to integrate modules for extraction-free sample lysis, loop-mediated isothermal amplification (LAMP) with lyophilized reagent beads, and real-time colorimetric signal sensing into a single miniaturized device, considerably shortening the sample-to-result time to 15 min. The precision liquid handling in the completely sealed microfluidic chip is achieved by a uniquely designed pressure-balanced automatic liquid flow mechanism, thereby eliminating the need for manual manipulation of liquids and thus the risk of biohazards. The PAD employs an improved real-time colorimetric LAMP (rcLAMP) assay with a limit of detection (LOD) of 1 copy/μL, enabled by enhanced assay chemistry to maximize the reaction kinetics. To validate this device for clinical application, we tested 206 clinical cervical swab samples and obtained a sensitivity of 92.1% and a specificity of 99.0%. This custom PAD enabled by microfluidic and electronic engineering techniques can be configured for the simultaneous detection of HPV16 and HPV18 or other pathogens in point-of-care applications.

Rapid detection of Japanese eel endothelial cell-infecting virus (JEECV) and Anguillid herpesvirus (AngHV-1) in Japanese eel (Anguilla japonica) using a qLAMP assay

Two DNA viruses, Japanese eel endothelial cell-infecting virus (JEECV) and Anguillid herpesvirus 1 (AngHV-1), cause serious diseases in eels that can negatively affect the economy of eel aquaculture industries in South Korea. Efficient virus detection is crucial for disease management; however, the practical application of current methodologies is challenging, in part owing to extensive time and specialized equipment requirements. The aim of this study was to develop a rapid real-time quantitative loop-mediated isothermal amplification (qLAMP) technique for the detection of JEECV and AngHV-1. Six different primers were designed to target eight different regions of the large T-antigen gene for JEECV detection, and the DNA polymerase catalytic subunit gene for AngHV-1 detection. Both sets of primers worked optimally at 65 °C. The qLAMP assay exhibited a minimum detection limit of 2.39 × 104 copies/μL for JEECV and 2.37 × 104 copies/μL for AngHV-1. Moreover, the primers demonstrated excellent specificity, showing no reaction with two major viruses, namely ostreid herpesvirus-1 microvariant (OsHV-1 μvar) and red sea bream iridovirus (RSIV). In addition, JEECV primers did not react with AngHV-1, and vice versa. Based on the standard curve and coefficient of determination values, both viruses could be quantified using this method. The qLAMP assay was validated with 104 clinical samples, including 72 infected and 32 healthy eels, and the assay findings were compared with previously reported duplex qPCR results that reported high sensitivity and specificity. The qLAMP results concorded well with the previous results. Evaluation of the threshold time (Tt) of the qLAMP results revealed that the samples could be analyzed in less than 15 min. The clinical diagnostic sensitivity (dSe) and diagnostic specificity (dSp) were assessed based on the confusion matrix; dSe and dSp for JEECV were 100% and 94.1%, respectively, and dSe and dSp for AngHV-1 were 96.8% and 77.8%, respectively. The Kappa values, which were used to represent the level of agreement between qLAMP and duplex qPCR results, for JEECV and AngHV-1 were 0.956 and 0.755, respectively, indicating that the qLAMP assay is a promising detection method for those viruses. Thus, the qLAMP assay developed herein could be applied as a rapid and sensitive tool to diagnose diseases caused by JEECV and AngHV-1 in the eel aquaculture industry.

Real-time loop-mediated isothermal amplification (real-time LAMP) assay for rapid diagnosis of amoebic liver abscess.

Among the parasitic diseases, amoebic liver abscess (ALA) ranks second to malaria in terms of mortality. Due to the poor sensitivity of conventional diagnostic methods, there is a need for the development of effective and rapid diagnostic methods for ALA. Thus, the purpose of this work was to develop a real-time loop-mediated isothermal amplification (RT-LAMP) assay specific to Entamoeba histolytica. Further, we compared the performance of real-time LAMP with conventional and real-time PCR (RT-PCR) targeting 18S small subunit ribosomal RNA (18S SSU rRNA) gene of E. histolytica in patients with ALA. A total of 126 liver samples were obtained for the study. Of these, 96 aspirated pus samples were obtained from patients suffering from an ALA (serology confirmed, anti-amoebic immunoglobulin IgG positive), 19 aspirated pus samples from patients with pyogenic liver abscess (PLA, 16S RNA gene positive) and 11 autopsy liver tissues. The results showed that the DNA of E. histolytica was detected in 81 samples by conventional PCR, 93 by RT-PCR and 95 by RT-LAMP. The analytical sensitivity of the RT-LAMP assay was much higher than the other two techniques. RT-LAMP assay was able to amplify up to one copy of the targeted gene of E. histolytica while conventional PCR and RT-PCR could amplify up to 103 and 102 copies of the targeted gene of E. histolytica, respectively. In conclusion, RT-LAMP proved to be a sensitive, specific and rapid test which can be utilised as an effective tool for the diagnosis of ALA.

Development and evaluation of an easy to use real-time reverse-transcription loop-mediated isothermal amplification assay for clinical diagnosis of West Nile virus

West Nile Virus (WNV) causes a serious public health concern in many countries around the world. Virus detection in pathological samples is a key component of WNV infection diagnostic, classically performed by real-time PCR. In outbreak situation, rapid detection of the virus, in peripheral laboratories or at point of care, is crucial to guide decision makers and for the establishment of adequate action plans to prevent virus dissemination. Here, we evaluate a Loop-mediated isothermal amplification (LAMP) tool for WNV detection. Amplifications were performed comparatively on extracted viral RNA and on crude samples using a classical thermal cycler and a portable device (pebble device). qRT-PCR was used as gold standard and two sets of urine samples (n = 62 and n = 74) were used to evaluate the retained amplification protocols and assess their sensitivity and specificity. RT-LAMP on RNA extracts and crude samples showed a sensitivity of 90 % and 87 %, respectively. The specificity was 100 % for extracts and 97 % for crude samples. Using the device, the RT-LAMP on extracted RNA was comparable to the gold standard results (100 % sensitivity and specificity) and it was a bit lower on crude samples (65 % sensitivity and 94 % specificity). These results show that RT-LAMP is an efficient technique to detect WNV. RT-LAMP provides a rapid, sensitive, high-throughput and portable tool for accurate WNV detection and has potentials to facilitate diagnostic and surveillance efforts both in the laboratory and in the field, especially in developing countries.

A real-time colourimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of highly pathogenic H5 clade 2.3.4.4b avian influenza viruses

ABSTRACT Highly pathogenic avian influenza viruses (HPAIV) are a major threat to the global poultry industry and public health due to their zoonotic potential. Since 2016, Europe and France have faced major epizootics caused by clade 2.3.4.4b H5 HPAIV. To reduce sample-to-result times, point-of-care testing is urgently needed to help prevent further outbreaks and the propagation of the virus. This study presents the design of a novel real-time colourimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of clade 2.3.4.4b H5 HPAIV. A clinical validation of this RT-LAMP assay was performed on 198 pools of clinical swabs sampled in 52 poultry flocks during the H5 HPAI 2020–2022 epizootics in France. This RT-LAMP assay allowed the specific detection of HPAIV H5Nx clade 2.3.4.4b within 30 min with a sensitivity of 86.11%. This rapid, easy-to-perform, inexpensive, molecular detection assay could be included in the HPAIV surveillance toolbox.

Compact Point-of-Care Device for Self-Administered HIV Viral Load Tests from Whole Blood.

Human immunodeficiency virus (HIV) is a significant problem to consider as it can lead to acquired immune deficiency syndrome (AIDS). Fortunately, AIDS is manageable through antiretroviral therapy (ART). However, frequent viral load monitoring is needed to monitor the effectiveness of the therapy. The current reverse transcription-polymerase chain reaction (RT-PCR) viral load monitoring is highly effective, but is challenged by being resource-intensive and inaccessible, and its turnaround time does not meet demand. An unmet need exists for an affordable, rapid, and user-friendly point-of-care device that could revolutionize and ensure therapeutic effectiveness, particularly in resource-limited settings. In this work, we explored a point-of-care HIV viral load device to address this need. This device can perform streamlined plasma separation, viral RNA extraction, and real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) semiquantitative testing in an ultracompact device. We developed an absorption-based membrane plasma separation method suitable for finger-prick blood samples, achieving an efficiency of 80%. We also designed a syringe-based RNA extraction method for on-site plasma processing with a viral recovery efficiency of 86%. We created a portable device with a smartphone interface for real-time semiquantitative RT-LAMP, which is useful for monitoring viral load. The device uses lyophilized reagents, processed with our lyophilization method, which remain stable for 16 weeks. The device can accurately categorize viral load into low, medium, and high categories with 95% accuracy. We believe this point-of-care HIV self-test device, offering convenience and long-term storage, could aid patients in home-based ART treatment monitoring.

Advancing Multiple Detection in RT-LAMP with a Specific Probe Assembled from Plural Three-Way-Junction Structures.

The timely detection of diseases and the accurate identification of pathogens require the development of efficient and reliable diagnostic methods. In this study, we have developed a novel specific multivariate probe termed MRTFP (multivariate real-time fluorescent probe) by assembling strand exchange three-way-junction (3WJ) structures. The 3WJ structures were incorporated into a four-angle probe (FP) and a hexagonal probe (HP), to target the multivariate genes of Salmonella. The FP and HP enable single-step and multiplexed detection in RT-LAMP (real-time loop-mediated isothermal amplification) with exceptional sensitivity and specificity. Encouragingly, real food samples contaminated with Salmonella (Salmonella enteritidis and Salmonella typhimurium) can be readily identified and distinguished with a minimum detectable concentration (MDC) of 103 CFU/mL without the need for further culture. The introduction of MRTFP allows for simultaneous detection of dual or three targets in a single tube for LAMP, thereby improving detection efficiency. The MRTFP simplifies the design of robust multivariate probes, exhibits excellent stability, and avoids interference from multiple probe units, offering significant potential for the development of specific probes for efficient and accurate disease detection and pathogen identification.

Real-Time and Rapid Detection of Phytopythium vexans Using Loop-Mediated Isothermal Amplification Assay.

Phytopythium vexans (de Bary) Abad, de Cock, Bala, Robideau, A. M. Lodhi & Levesque is an important waterborne and soil-inhabiting oomycete pathogen causing root and crown rot of various plants including certain woody ornamentals, fruit, and forest trees. Early and accurate detection of Phytopythium in the nursery production system is critical, as this pathogen is quickly transported to neighboring healthy plants through the irrigation system. Conventional methods for the detection of this pathogen are tedious, frequently inconclusive, and costly. Hence, a specific, sensitive, and rapid molecular diagnostic method is required to overcome the limitations of traditional identification. In the current study, loop-mediated isothermal amplification (LAMP) for DNA amplification was developed for the identification of P. vexans. It was evaluated using real-time and colorimetric assays. Several sets of LAMP primers were designed and screened, but PVLSU2 was found to be specific to P. vexans as it did not amplify other closely related oomycetes, fungi, and bacteria. Moreover, the developed assays were sensitive enough to amplify DNA up to 102 fg per reaction. The real-time LAMP assay was more sensitive than traditional PCR and culture-based methods to detect infected plant samples. In addition, both LAMP assays detected as few as 100 zoospores suspended in 100 ml water. These LAMP assays are anticipated to save time in P. vexans detection by disease diagnostic laboratories and research institutions and enable early preparedness in the event of disease outbreaks.

Real-time loop-mediated isothermal amplification for the detection of Giardia duodenalis in fecal specimens

IntroductionGiardiasis is a leading cause of subacute or chronic diarrhoea and is frequently associated with impaired physical, cognitive and psychosocial development, especially in children. The diagnosis relies mainly on the microscopic evaluation of stool specimens that have a low sensitivity. In contrast, molecular advancements like the polymerase chain reaction and Real-time loop-mediated isothermal amplification (Real-time LAMP) are promising techniques and reportedly have better diagnostic characteristics. MethodsWe have evaluated the performance of Real-time LAMP for detecting Giardia in ninety stool specimens compared to microscopy and nested PCR. ResultsA total of 35 fecal samples were detected positive by microscopy, 41 by nested PCR and 43 by real-time LAMP. Microscopy and nested PCR detected 33, microscopy and real-time LAMP detected 35, and nested PCR and real-time LAMP detected 41 positive samples. ConclusionThe real-time LAMP assay was found suitable for the rapid and accurate detection of G. duodenalis with a better sensitivity in comparison to nested PCR and microscopy. Furthermore, besides being sensitive and rapid, LAMP had the advantage of an adequate rapid turn-around time of eleven to 15 ​min as compared to 5 ​h of nested PCR.

Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Ralstonia solanacearum Species Complex and Phylotype I in Solanaceous Crops

The Ralstonia solanacearum species complex (RSSC) causes bacterial wilt diseases, which affect a wide range of plant hosts, including Solanaceae, and is of major economic importance. Point-of-care (POC) techniques such as loop-mediated isothermal amplification (LAMP) enable the rapid and sensitive detection of plant pathogens and can be deployed directly in the field. For fast and reliable diagnosis on site, we optimized an RSSC-LAMP assay and developed a real-time LAMP assay targeting the phylotype I. Both LAMPs were highly specific, yielding negative results for a wide collection of nontarget strains and positive results for all target strains, except for two particular mulberry strains for the phylotype I assay. These results were supported by an extensive in silico analysis performed on 6,105 genomes of Burkholderiaceae. The two LAMP assays displayed high sensitivity on pure suspensions, with a detection limit at 103 and 104 CFU/ml for phylotype I and the RSSC, respectively. These thresholds correspond to theoretical quantities of as low as 5 and 50 CFU per reaction. Two simplified extraction methods were successfully used to detect the pathogen from different Solanaceae samples. We demonstrated that LAMP assays were operable on solanaceous crops during field surveys and varietal evaluation trials as rapid POC diagnostics tools, which can improve the disease management and control of the RSSC. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .