Positive Loop-mediated Isothermal Amplification Research Articles

Loop-mediated isothermal amplification (LAMP) assay coupled with gold nanoparticles for colorimetric detection of Trichoderma spp. in Agaricus bisporus cultivation substrates

One of the significant challenges in organic cultivation of edible mushrooms is the control of invasive Trichoderma species that can hinder the mushroom production and lead to economic losses. Here, we present a novel loop-mediated isothermal amplification (LAMP) assay coupled with gold nanoparticles (AuNPs) for rapid colorimetric detection of Trichoderma spp. The specificity of LAMP primers designed on the tef1 gene was validated in silico and through gel-electrophoresis on Trichoderma harzianum and non-target soil-borne fungal and bacterial strains. LAMP amplification of genomic DNA templates was performed at 65 °C for only 30 min. The results were rapidly visualized in a microplate format within less than 5 min. The assay is based on salt-induced aggregation of AuNPs that is being prevented by the amplicons produced in case of positive LAMP reaction. As the solution color changes from red to violet upon nanoparticle aggregation can be observed with the naked eye, the developed LAMP-AuNPs assay can be easily operated to provide a simple initial screening for the rapid detection of Trichoderma in button mushroom cultivation substrate.

Detection of respiratory pathogenic bacterial nucleic acid detection by Loop-mediated Isothermal Amplification in patients with bacterial pulmonary infections

ObjectiveNucleic acid testing can accurately and rapidly identify the presence of pathogenic bacteria. In this study, we analyzed respiratory pathogenic bacteria nucleic acids by LAMP (Loop-mediated isothermal amplification) to clarify the clinical application in patients with bacterial pulmonary infections. MethodsClinical data and specimens were collected from 99 patients with bacterial pulmonary infections from June 2021 to April 2023. We compared the differences between nucleic acid detection of LAMP and sputum culture. The correlation between inflammation manifestations of pulmonary imaging and the nucleic acid detection of LAMP was compared and analyzed. And the relationship between LAMP and blood inflammatory markers were analyzed. ResultsThe positive rate of LAMP using sputum specimens was significantly higher than that of sputum culture (P < 0.05). Pathogenic bacteria in sputum samples are more likely to be detected by LAMP in patients with inflammatory on lung imaging examination. The coincidence rate of elevated PCT and CRP expression with positive LAMP results were 83.87 % and 88.71 %, respectively. Moreover, PCT, CRP and WBC were significantly higher in LAMP positive group than those in negative group (P < 0.05). ConclusionNucleic acid testing of sputum specimens for pathogenic bacteria by LAMP on the basis of imaging examination can provide a rapid and accurate experimental basis for clinical diagnosis of bacterial pulmonary infections.

Analysis of the factors that affect the detection duration of SARS-CoV-2 in Loop Mediated Isothermal Amplification among COVID-19 inpatients.

In COVID-19 patients who are severe or immunocompromised, the duration of infectious viral shedding may be longer, and a longer isolation duration is recommended. In National Sagamihara hospital, a decline in the viral load to end the isolation of COVID-19 hospitalized patients is confirmed by loop mediated isothermal amplification (LAMP). However, a subset of patients persisted in displaying LAMP positivity for more than 20 days since symptom onset. Therefore, we conducted a retrospective observational study to investigate factors impacting the persistence of LAMP positivity. The study included 102 participants. The severity of COVID-19 was mild in 25.5%, moderate in 67.6%, and severe in 6.9% of patients. The median number (interquartile range) of days until negative LAMP since symptom onset was 16 (14-19) days. Multivariate logistic regression analysis showed that age ≥55 years and the delta variant were correlated with persistently LAMP positive for more than 20 days since symptom onset. This study identified that age, the delta variant, and oxygen requirement were factors contributing to persistently positive LAMP. Therefore, it is posited that in these patients, the implementation of LAMP for de-isolation would result in a prolonged duration of isolation.

Development of a loop-mediated isothermal amplification (LAMP)-based electrochemical test for rapid detection of SARS-CoV-2

Rapid, reliable, sensitive, portable, and accurate diagnostics are required to control disease outbreaks such as COVID-19 that pose an immense burden on human health and the global economy. Here we developed a loop-mediated isothermal amplification (LAMP)-based electrochemical test for the detection of SARS-CoV-2 that causes COVID-19. The test is based on the oxidation-reduction reaction between pyrophosphates (generated from positive LAMP reaction) and molybdate that is detected by cyclic voltammetry using inexpensive and disposable carbon screen printed electrodes. Our test showed higher sensitivity (detecting as low as 5.29 RNA copies/μL) compared to the conventional fluorescent reverse transcriptase (RT)-LAMP. We validated our tests using human serum and saliva spiked with SARS-CoV-2 RNA and clinical (saliva and nasal-pharyngeal) swab samples demonstrating 100% specificity and 93.33% sensitivity. Our assay provides a rapid, specific, and sensitive test with an electrochemical readout in less than 45min that could be adapted for point-of-care settings.

Phenol red as electrochemical indicator for highly sensitive quantification of SARS-CoV-2 by loop-mediated isothermal amplification detection

The current COVID-19 pandemic has made patent the need for rapid and cost-effective diagnostic tests, crucial for future infectious outbreaks. Loop-mediated isothermal amplification (LAMP) is a promising and decentralized alternative to qPCR. In this work we have developed a sensitive, fast, and simple innovative methodology for quantification of SARS-CoV-2 RNA copies, combining reverse-transcription LAMP with electrochemical detection. This is based on the oxidation of phenol red (PR), a visual and electroactive LAMP indicator, which oxidation peak potential (Ep) changes with the progress of the LAMP reaction. Using that Ep shift as analytical signal, a calibration curve was obtained for fragment N1 copies of SARS-CoV2 (which provided better results than N or S fragments), with a potential shift of 16.2 mV per order of magnitude, and a practical limit of detection of 21 copies·μL−1. Moreover, the precision of Ep is excellent (RSD < 2%): 557 ± 5 mV for negative and 602 ± 7 mV for positive (2148 N fragment RNA copies·µL-1·-1) LAMP controls. This methodology has been applied to the analysis of nasopharyngeal swab samples, resulting in total concordance with clinical RT-qPCR results. Advances towards fully decentralization have been achieved by designing and fabricating a small portable heater for isothermal procedures, obtaining comparable results to those from a commercial benchtop thermal cycler.

Development of a Loop-Mediated Isothermal Amplification (LAMP) Method to Detect the Potato Zebra Chip Pathogen 'Candidatus Liberibacter solanacearum' (Lso) and Differentiate Haplotypes A and B.

'Candidatus Liberibacter solanacearum' (Lso) is the causal agent of zebra chip of potato (Solanum tuberosum), which can significantly reduce potato yield. In this study, a loop-mediated isothermal amplification (LAMP) method for the detection of Lso haplotypes A and B was developed and evaluated. Two sets of LAMP primers named LAMP-A and LAMP-B were designed and tested for specificity and sensitivity. Both LAMP-A and LAMP-B were specific to Lso in in silico analysis using the Primer-Blast tool. The LAMP-A and LAMP-B could only produce positive signals from DNA mixtures of Lso-infected tomato but not from the genomic DNA of 37 nontarget plant pathogens. The sensitivity of LAMP-A and LAMP-B on Lso haplotypes A and B were tested on gBlocks and genomic DNA from Lso-infected tomato. On the genomic DNA for LAMP-A, the lowest amount of template DNA for a positive LAMP reaction was 2 to 20 ng on four haplotype A strains and 20 to 80 ng on four haplotype B strains; for LAMP-B, the lowest amount of template DNA for a positive LAMP reaction was 0.02 to 2 ng on four haplotype B strains and 20 ng to no amplification on four haplotype A strains. On gBlocks for LAMP-A, the lowest number of copies for a positive LAMP reaction was 60 on haplotype A and 600 on haplotype B; for LAMP-B, the lowest number of copies for a positive LAMP reaction was 60 on haplotype B and 600 on haplotype A. Therefore, considering the convenience of the LAMP technique, as well as the high specificity and sensitivity, the LAMP-A and LAMP-B primers can be used together to test the probable Lso-infected plant or psyllid samples to rapidly, accurately, and directly differentiate haplotypes A and B. We highly recommend this LAMP system to plant pathology practitioners and diagnostic labs for routine detection of Lso and confirmation of zebra chip disease on potato or tomato.

Development and Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Method for Detection of the Potato Powdery Scab Pathogen Spongospora subterranea.

Spongospora subterranea is the causal agent of powdery scab of potato (Solanum tuberosum), which can significantly reduce potato quality. In this study, we developed and evaluated a loop-mediated isothermal amplification (LAMP) method for the detection of S. subterranea. A set of LAMP primers named PS-LAMP was designed and tested for specificity and sensitivity. In the specificity test, in silico analysis using the NCBI Primer-BLAST tool indicated that PS-LAMP was specific to S. subterranea. The in vitro tests confirmed specificity, showing that PS-LAMP could produce positive signals from DNA isolated from each of three potato tubers with powdery scab symptoms but did not produce positive signals from DNA isolated from 38 nontarget plant pathogens. The sensitivity of PS-LAMP was tested on both gBlocks and DNA isolated from potato samples with powdery scab symptoms. On gBlocks, the lowest number of copies for a positive LAMP reaction was six, which was similar to results obtained via qPCR, but it was 10 times more sensitive than conventional PCR. On a DNA sample from S. subterranea-infected potato, the lowest amount of template DNA for a positive LAMP reaction was 2 pg, which was incomparable with the sensitivity of qPCR. Considering the convenience of the LAMP technique, as well as the high specificity and sensitivity, this assay can be very useful for plant pathology practitioners and diagnostic labs interested in rapid, accurate, and routine detection of S. subterranea and confirmation of powdery scab disease.

Amine-Functionalized Quantum Dots as a Universal Fluorescent Nanoprobe for a One-Step Loop-Mediated Isothermal Amplification Assay with Single-Copy Sensitivity.

Loop-mediated isothermal amplification (LAMP) has received considerable attention for decentralized (point-of-care and on-site) nucleic acid testing in view of its simple temperature control (60-65 °C) and short assay time (15-60 min). There remains a challenge in its wide adoption and acceptance due to the limitations of the existing amplification result reporter probes, e.g., photobleaching of organic fluorophore and reduced sensitivity of the pH-sensitive colorimetric dye. Herein, we demonstrate CdSeS/ZnS quantum dots (semiconductor fluorescent nanocrystals with superior photostability than organic fluorophore) with surface modification of cysteamine (amine-QDs) as a new reporter probe for LAMP that enabled single-copy sensitivity (limit of detection of 83 zM; 20 μL reaction volume). For a negative LAMP sample (absence of target sequence), positively charged amine-QDs remained dispersed due to interparticle electrostatic repulsion. While for a positive LAMP sample (presence of target sequence), amine-QDs became precipitated. The characterization data showed that amine-QDs were embedded in magnesium pyrophosphate crystals (generated during positive LAMP), thus leading to their coprecipitation. This amine-QD-based one-step LAMP assay advances the field of QD-based nucleic acid amplification assays in two aspects: (1) compatibility─one-step amplification and detection (versus separation of amplification and detection steps); and (2) universality─the same amine-QDs for different target sequences (versus different oligonucleotide-modified QDs for different target sequences).

Development of a photothermal bead-based nucleic acid amplification test (pbbNAAT) technique for a high-performance loop-mediated isothermal amplification (LAMP)–based point-of-care test (POCT)

We have developed a novel molecular diagnostic platform (photothermal bead-based nucleic acid amplification test; pbbNAAT) that greatly improves the low sensitivity of direct loop-mediated isothermal amplification (LAMP) and allows for specific detection of LAMP amplicons in complex samples. The pbbNAAT integrates specific ligand-functionalized polypyrrole-coated iron oxide particles (PPy@IOs) capable of photothermal conversion and single-molecule magnetic capture of target analytes, the released nucleic acid, and LAMP-amplified products under external light energy control and magnetic manipulations. This allows for sample pretreatment, pbbLAMP amplification, and subsequent amplicon detection with bead-based ELISA in a one-stop microreactor without loss. In addition, photonic heating with PPy@IOs and external light control provide instant and uniform heating for thermolysis and pbbLAMP implementations. Moreover, it generates higher primer annealing stringency for LAMP primers in pbbLAMP; thus, it can detect pathogen-specific DNA accurately and promptly in pathogen-spiked complex materials. The sample pretreatment procedure of pbbNAAT can greatly reduce inhibitors originating from complex samples, which enables the maintenance of maximal enzyme activities for highly sensitive detection. More importantly, the pbbLAMP assay coupled with magnetic capture permits subsequent bead-based ELISA detection to determine true positive LAMP amplicons on PPy@IOs. The pbbNAAT platform has a high tolerance to inhibitors originating from complex samples, high analytical specificity, and limitation of detection (LoD) as low as 8 CFU/reaction to detect E. coli spiked in human whole blood, bovine milk, and can be completed in less than 1 h. Therefore, we believe that pbbNAAT can serve as a suitable direct LAMP platform for on-site POCTs.

Micro-Probe Potentiometric pH Sensor for Detection of Amplification in the LAMP Assay for White Spot Syndrome Virus (WSSV) in Shrimps

A novel application of pH sensors is in the detection of amplification in nucleic acid-based methods for diagnosis of organisms. The detection is based on the production of hydrogen ions during the amplification process. In this study, a micro-probe potentiometric sensor for pH was fabricated and applied in the reagentless detection of the White Spot Shrimp Virus (WSSV) through the loop-mediated isothermal amplification (LAMP) technique. The pH micro-probe contained a pencil graphite modified with polypyrrole nanotubes through galvanostatic electrodeposition in the presence of methyl orange (MO). Electropolymerization parameters and potentiometric measurements parameters were optimized to obtain maximum sensitivity. The probe was characterized using Field Emission–Scanning Electron Microscopy (FE-SEM) and Fourier Transform-Infrared (FT-IR) Spectroscopy. The sensor showed a sub-Nernstian response (−45.5 mV/pH) at pH range 2–8 and LOD of <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">${5.19}\times {10}^{-{8}}$ </tex-math></inline-formula> M; with minimum hysteresis ( <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\Delta $ </tex-math></inline-formula> m = ± 1.27 mV/pH), and low potential drift (±1.00 mV, pH 8). The probe was used to measure the pH of the microliter-volume reaction mixture at the start and at the end of the LAMP assay for WSSV. The probe detected pH change ( <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\Delta $ </tex-math></inline-formula> pH = −0.675) that occurred during the LAMP assay. This behavior correlated well with the results of fluorescence detection of the LAMP assay kit using SYBR Safe® DNA gel stain. A high diagnostic sensitivity (93.8%) and specificity (100%) was observed. The fabricated potentiometric sensor showed promising result in monitoring positive LAMP reaction through measurement of pH. This provides a method to diagnose shrimp samples with WSSV that is rapid, easy-to-operate, and cost-effective.

Performance of loop-mediated isothermal amplification (LAMP) assay for detection of Schistosoma japonicum infection in Oncomelania snails in schistosomiasis transmission-interrupted regions

To compare the effectiveness of loop-mediated isothermal amplification (LAMP) assay and microscopic examinations for detection of Schistosoma japonicum infections in Oncomelania hupensis in transmission-interrupted regions, so as to provide insights into the optimization of snail surveillance tools in these regions. Four hilly schistosomiasis-endemic villages where transmission interruption was achieved were selected in Heqing County of Yunnan Province as the study villages, including Xinzhuang and Gule villages in hilly regions and Lianyi and Yitou villages in dam regions. Snail survey was performed by means of systematic sampling combined with environmental sampling in July 2018. All captured snails were identified for S. japonicum infections using microscopy. In addition, 10 to 20 snails were randomly sampled from each snail habitat following microscopy, numbered according to environments and subjected to LAMP assay. The positive rate of settings with S. japonicum-infected snails was compared among villages. A total of 7 949 living snails were captured from 83 snail habitats in 4 villages, and no S. japonicum infection was detected in snails. There were 226 mixed samples containing 1 786 snails subjected to LAMP assay, and positive LAMP assay was found in 3 mixed samples from 3 snail habitats in 2 dam villages. The positive rates of settings with S. japonicum-infected snails were comparable between Lianyi Village (one setting) and Yitou Village (2 set tings) (5.89% vs. 14.29%, P = 0.344). However, the overall positive rate of settings with S. japonicum-infected snails was significantly higher in dam villages (9.67%, 3/31) than in hilly villages (0) (P = 0.048). LAMP assay is more sensitive to detect S. japonicum infections in O. hupensis than conventional microcopy method, which may serve as a supplementary method for detection of S. japonicum infections in O. hupensis in high-risk snail habitats in hilly transmission-interrupted regions.

Rapid identification of Atlantic salmon (Salmo salar) based on loop-mediated isothermal amplification (LAMP) using self-quenching fluorogenic approach

Loop-mediated isothermal amplification (LAMP) has emerged as an important diagnostic tool for fish authenticity purpose. While the sequence-independent detection methods greatly increased the likelihood of detecting false-positive signals. Instead, target-specific detection of LAMP amplicons can be achieved through the use of target-specific probes or modified primers as biorecognition elements. The present work selected Salmo salar as a case study, and developed a novel LAMP assay coupled with the self-quenching approach for accurate species identification.Specifically, the loop primer (LB-6) was identified as the candidate primer to design the self-quenching element. The fluorophore attached to LB-6 is self-quenched in unbound state. After binding to the dumb bell shaped DNA specifically, the FAM fluorophore is de-quenched, resulting in the fluorescence release. With the novel assay, as little as 5 pg of S. salar DNA could be detected. Moreover, the self-quenching assay embraces a high tolerance to impurities and the positive LAMP results can always be obtained, regardless of the DNA extraction methods and sample treatments. While significant difference with the Ct value according to both the extraction methods and sample treatments, highlighted also an improved LAMP amplification using DNA with higher purity.

A direct multiplex loop‐mediated isothermal amplification method to detect three CITES‐listed shark species

Abstract Species identification of sharks under catch or trade regulations is important for law enforcement and species conservation. Rapid detection of Convention on the International Trade in Endangered Species (CITES)‐listed species is needed for on‐site screening. Species‐specific primers were designed to target three mitochondrial genes (ND2, COI, and CytB) in both the simplex and multiplex loop‐mediated isothermal amplification (LAMP) assay for the pelagic thresher shark (Alopias pelagicus), the bigeye thresher shark (Alopias superciliosus), and the scalloped hammerhead shark (Sphyrna lewini), respectively. Another primer set designed to target S. lewini was used for detection‐limit testing of the LAMP assay. The refined direct multiplex LAMP was used to detect the three CITES‐listed shark species and omitted the lengthy DNA extraction process. A homogenizer was used to release the DNA from the shark tissues, and a simplex or multiplex LAMP reaction was conducted for 30 min in an incubator at 65°C using species‐specific primer sets. Positive LAMP reactions showed a colour change from pink to yellow, whereas negative reactions showed no colour change. Multiplex LAMP assays were performed using 84 samples, which successfully identified the target and non‐target species and provided a fast (&lt;1 h), simple, and reliable method to distinguish three CITES‐listed shark species from the other non‐target species, for either fresh or dry fin products. Results of this study and the method developed will play a critical role in assisting fishery agencies and customs officials in identifying the illegal catch and trade of CITES‐listed shark species.

A diagnostic LAMP assay for the destructive grapevine insect pest, phylloxera (Daktulosphaira vitifoliae)

Grape phylloxera (Daktulosphaira vitifoliae) is a destructive insect pest of grapevines that is highly invasive worldwide, despite strict biosecurity containment measures in place at farm and regional levels. Current phylloxera identification by visual inspection and laboratory-based molecular methods is time-consuming and costly. More rapid and cost-effective methods for identification of this pest would benefit industry, growers, and biosecurity services. Loop mediated isothermal amplification (LAMP) is a new portable technology available for rapid and accurate in-field molecular diagnostics. This study outlines the development of a new LAMP assay to enable the identification of phylloxera specimens. New LAMP primers were developed to specifically amplify phylloxera mitochondrial DNA (5′-COI), which we have shown is effective as a DNA barcode for identification of phylloxera, using LAMP technology. Positive LAMP reactions, containing phylloxera DNA, amplified in less than twelve minutes with an anneal derivative temperature of approximately 79 °C to 80 °C compared to a newly designed synthetic DNA (gBlock) fragment which had an anneal derivative temperature of 82 °C. No LAMP amplification was detected in any of the non-target species tested, i.e. no false-positive identification resulted for these species. We also successfully optimised a non-destructive DNA extraction procedure, HotSHOT “HS6”, for use in the field on phylloxera adults, nymphs and eggs, to retain physical specimens. DNA extracted using this method was also suitable for species and genotype molecular identification methods, such as DNA barcoding, qPCR and microsatellite genotyping. The new LAMP assay provides a novel visual molecular tool for accurate diagnostics of phylloxera in the laboratory and field.

Development of a loop-mediated isothermal amplification assay for rapid detection of African swine fever

Since the first African swine fever (ASF) outbreak was reported in China in 2018, the disease has spread rapidly to several countries in Asia. The early detection of this disease is essential for the ASF control strategy to be effective. Loop-mediated isothermal amplification (LAMP) is a nucleic acid detection assay that is rapid, simple, cost-effective and field-friendly. In this study, we have developed a colorimetric assay of LAMP to detect ASF virus (ASFV). A set of LAMP primers was designed to target the conserved region of the VP72 gene. The conditions of LAMP were optimized. The amplification products were easily detected by the naked eye using hydroxynaphthol blue (HNB). The positive LAMP reaction generated a violet to sky blue color change. The sensitivity and specificity of LAMP assay were demonstrated in comparison with the OIE-recommended real-time PCR. A total of 211 samples including 121 confiscated pork products and 90 spiked clinical specimens were tested. The optimal amplification of ASFV DNA by LAMP was incubation at 60 °C for 90 min. The analytical sensitivity of ASFV LAMP assay was at least 368 plasmid DNA copies/µL without cross-reactivity with other swine pathogens. The diagnostic sensitivity and specificity of LAMP were 88% and 100%, respectively. There was almost perfect agreement between LAMP and real-time PCR assays (Kappa value=0.84). This novel LAMP assay is deemed to be a rapid, simple, sensitive, specific diagnostic tool and suitable for early detection of ASF to minimize the likelihood of ASF spread nationwide.

Rapid diagnosis of Theileria annulata by colorimetric loop-mediated isothermal amplification (LAMP) assay

Theileria annulata the causative agent of bovine tropical theileriosis (BTT) is globally distributed. Rapid and accurate detection of the parasite is essential for the implementation and evaluation of mass drug administration and planned vaccination programs for controlling BTT. Loop mediated isothermal amplification (LAMP) amplifies targeted nucleic acid with a high efficacy, sensitivity and rapidity under isothermal conditions. In the present study, the internal transcriber space (ITS) gene of T. annulata was targeted for the development of a LAMP assay using pH-sensitive dye (phenol red) for enhanced visual detection of amplification. This method employed a set of specially designed four primers that recognized six distinct regions on the targeted gene. No amplification was detected with the DNA of other tested haemoprotozoans. Positive LAMP products were identified by a colour change from pink to yellow, and then rechecked by specific ladder pattern upon agarose gel electrophoresis. LAMP was able to detect infection in 63 out of 85 animals compared with blood microscopy, simple PCR and nested PCR which detected infection in 40, 49 and 64 animals, respectively. No difference in detection was seen in the colorimetric assay and the classical UV based LAMP assay.

Loop-mediated isothermal amplification for the early diagnosis of invasive meningococcal disease in children

BackgroundRapid molecular diagnostic testing has the potential to improve the early recognition of meningococcal disease (MD). The aim of this study was to report on the diagnostic test accuracy of...

Rapid identification and detection of Vibrio parahaemolyticus via different types of modus operandi with LAMP method in vivo

PurposeVibrio parahaemolyticus, an easy-ignored food-borne pathogen, can cause bacterial outbreaks and human disease during early-stage infection. In this study, we aimed to evaluate the detection efficiency of loop-mediated isothermal amplification (LAMP) as an emerging technique to directly detect V. parahaemolyticus infection in mammalian hosts and assess its potential in clinical applications.MethodsA LAMP assay was used for rapid identification of V. parahaemolyticus in a variety of mouse models in which animals were infected via the digestive tract, wounds, or through general infection, and the results were compared with routine analytical methods.ResultsOur results confirmed that the LAMP assay was capable of detecting V. parahaemolyticus in different mouse organs independent of the source of bacteria, although its sensitivity depended on the route of infection and the organ affected. Foodborne-derived V. parahaemolyticus was the most sensitive route, with the small intestine being the most sensitive organ. The LAMP assay indicated that V. parahaemolyticus that spread through the blood stream had the most serious consequences during early-stage infection. Positive LAMP results were identified in all blood samples from i.v. injected mice. Furthermore, the LAMP method could directly detect trace quantities of V. parahaemolyticus in fresh peripheral blood while conventional methods failed to do so, thereby shortening the time-to-result from days to minutes.ConclusionsIn this study, we demonstrated that the LAMP assay was effective in speeding up the detection of V. parahaemolyticus. Instead of being a secondary method to assist in the clinic, the LAMP assay has potential for use as the primary technique for rapid detection of V. parahaemolyticus in the future.

Tritrichomonas Foetus: A Study of Prevalence in Animal Hosts in Poland.

Tritrichomonas foetus is described as a pathogen of cattle and cats and also exhibits commensalism with pigs. In order to estimate the prevalence and determine the risk factors for parasite infection, specimens from animal hosts (cat, pigs, and cattle) from Poland were investigated. To our best knowledge, this is the first such study to examine samples from wild boars (Sus scrofa) for the presence of T. foetus. Data were collected from 117 cats, 172 pigs, 236 wild boars, and 180 cattle. The sensitivity of T. foetus identification was increased by using two molecular assays: PCR and loop-mediated isothermal amplification (LAMP). The prevalence of feline tritrichomonosis was 20.51%, and statistically significant differences were obtained between groups of animals regarding age, breed, number of cats, diarrhea, and place of living. Positive PCR and LAMP results for T. foetus were estimated for 16.28% of pigs, and the obtained data were significantly correlated with age. Conversely, no significant differences were observed concerning the farm size factor. In our survey, no cases of bovine tritrichomonosis were found, which is consistent with the data from the other countries of the European Union. Similarly, all wild boar samples were also T. foetus-negative according to LAMP and PCR.

DNA detection of Paragonimus westermani: Diagnostic validity of a new assay based on loop-mediated isothermal amplification (LAMP) combined with a lateral flow dipstick

Paragonimus westermani (P. westermani) is widely spread in Asian countries and is one of the most important causative agents for lung fluke diseases. The prevention and control of Paragonimiaisis mainly depends on the accurate diagnosis and effective treatment. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay targeted to a portion of the Ty3/gypsy-like LTR retrotransposon (Rn1) sequence coupled with a lateral flow dipstick (LFD) for the rapid detection of P. westermani-specific amplicons. The positive LAMP products were biotin-labeled and hybridized with a fluorescein isothiocyanate-labeled probe which could be visually detected by LFD. No cross-reaction were observed with other parasitic pathogens including Trichinella spiralis, Anisakis simplex, Schistosoma japonicum and Gnathostoma spinigerum, but this LAMP assay could not distinguish P. westermani with Paragonimus skrjabini and Paragonimus heterotremus. The detection limit of the LAMP assay for P. westermani was 2.7 fg/µL, while that of PCR method was 27 fg/µL. LAMP method was applied to detect P. westermani genomic DNA in blood samples form experimental infected dogs, and results showed the parasite was detectable as early as week 2. LAMP-LFD assay applicability was successfully tested in dog blood samples collected from five cities (Wenzhou, Hangzhou, Huzhou, Jiaxing and Shaoxing) in Zhejiang province. In summary, the established LAMP-LFD assay targeted to the Rn1 sequence is a rapid and convenient method for specific detection of P. westermani.