Browse
Submit Data
Databases
API
Help

Dataset Information

16 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Detection of microcystin-producing cyanobacteria in water samples using loop-mediated isothermal amplification targeting mcyB gene.

ABSTRACT: Microcystin toxin-producing cyanobacteria are known to have harmful effects on humans and animals. We have developed a loop-mediated isothermal amplification (LAMP)-based detection method by targeting the microcystin synthetase B gene (mcyB), the gene responsible for the production of microcystin. The sensitivity of the method was found to be 1 fg per reaction, and it was 1000-fold higher than the conventional PCR. The LAMP method was able to amplify the target gene with a minimum amount of dNTP (0.4 mM), which further reduces the cost of reaction. The improved LAMP assay could detect the presence of the toxin-producing cyanobacteria in water samples within 2 h of time, which demonstrates the rapidness of the method. Freshwater samples were screened using the developed LAMP, and seven water samples collected from lakes and a bird sanctuary tested positive for mcyB gene harboring cyanobacteria, and negative in all other drinking waters. Hence, the developed LAMP could be a possible alternative to the existing molecular methods for screening for microcystin in environmental samples with greater sensitivity.

SUBMITTER: Ramya M

PROVIDER: S-EPMC6097961 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Rapid culture-independent loop-mediated isothermal amplification detection of antimicrobial resistance markers from environmental water samples.

Project description:Environmental water is considered one of the main vehicles for the transmission of antimicrobial resistance (AMR), posing an increasing threat to humans and animals health. Continuous efforts are being made to eliminate AMR; however, the detection of AMR pathogens from water samples often requires at least one culture step, which is time-consuming and can limit sensitivity. In this study, we employed comparative genomics to identify the prevalence of AMR genes within among: Escherichia coli, Klebsiella, Salmonella enterica and Acinetobacter, using publicly available genomes. The mcr-1, blaKPC (KPC-1 to KPC-4 alleles), blaOXA-48, blaOXA-23 and blaVIM (VIM-1 and VIM-2 alleles) genes are of great medical and veterinary significance, thus were selected as targets for the development of isothermal loop-mediated amplification (LAMP) detection assays. We also developed a rapid and sensitive sample preparation method for an integrated culture-independent LAMP-based detection from water samples. The developed assays successfully detected the five AMR gene markers from pond water within 1 h and were 100% sensitive and specific with a detection limit of 0.0625 μg/mL and 10 cfu/mL for genomic DNA and spiked bacterial cells, respectively. The integrated detection can be easily implemented in resource-limited areas to enhance One Health AMR surveillances and improve diagnostics.

| S-EPMC10128135 | biostudies-literature

Rapid and sensitive detection of human astrovirus in water samples by loop-mediated isothermal amplification with hydroxynaphthol blue dye.

Project description:BACKGROUND: The aim of this paper was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, sensitive and inexpensive detection of astrovirus. RESULTS: The detection limit of LAMP using in vitro RNA transcripts was 3.6 × 10 copies·?L?¹, which is as sensitive as the presently used PCR assays. However, the LAMP products could be identified as different colors with the naked eye following staining with hydroxynaphthol blue dye (HNB). No cross-reactivity with other gastroenteric viruses (rotavirus and norovirus) was observed, indicating the relatively high specificity of LAMP. The RT-LAMP method with HNB was used to effectively detect astrovirus in reclaimed water samples. CONCLUSIONS: The LAMP technique described in this study is a cheap, sensitive, specific and rapid method for the detection of astrovirus. The RT-LAMP method can be simply applied for the specific detection of astrovirus and has the potential to be utilized in the field as a screening test.

| S-EPMC3930758 | biostudies-literature

Development of a loop-mediated isothermal amplification assay for detection of <i>Austropeplea tomentosa</i> from environmental water samples.

Project description:Lymnaeid snails are key intermediate hosts for the development and survival of Fasciola spp., the causative agent of Fascioliasis which are economically important parasites infecting humans and livestock globally. The current control method for treating Fascioliasis is heavily reliant on anthelmintic drugs, particularly Triclabendazole (TCBZ) which has resulted in drug-resistant parasites and poses significant risk as there are no long-term efficacious alternatives available. Sustainable control measures at the farm level could include both parasite and snail control will play an important role in Fasciola spp. control and reduce the reliance on anthelmintic drugs. Implementation of such sustainable control measures requires effective identification of snails on the property however Lymnaeid snails are small and difficult to physically locate. Snail identification using an environmental DNA approach is a recent approach in which physically locating snails are not required. Austropeplea tomentosa, is the primary intermediate snail host for F. hepatica transmission in South-East Australia and we present an in-field loop-mediated isothermal amplification and water filtering method for the detection of A. tomentosa eDNA from water samples to improve current surveillance methods. This methodology is highly sensitive with a detection limit of 5 × 10- 6 ng/μL, detected in < 20 minutes, with cumulative sample preparation and amplification time under 1 hour. This proposed workflow could assist in monitoring areas to determine the risk of Fascioliasis infection and implement strategies to manage snail populations to ultimately reduce the risk of infection for humans and livestock.Supplementary informationThe online version contains supplementary material available at 10.1186/s44149-022-00061-9.

| S-EPMC9743122 | biostudies-literature

Rapid visual detection of binary toxin producing Clostridium difficile by loop-mediated isothermal amplification.

Project description:The binary toxin Clostridium difficile transferase (CDT) is frequently observed in C. difficile strains and is associated with an increased severity of C. difficile infection. CDT-producing C. difficile infections cause higher fatality rates than infections with CDT negative isolates. Thus, the rapid and accurate identification of a CDT positive C. difficile infection is critical for effective treatment. The present study demonstrates how loop-mediated isothermal amplification (LAMP) can be used to detect CDT-producing C. difficile based on visual observation. This is a low complexity, rapid molecular method that has the potential to be used within a point of care setting. The specificity and sensitivity of the primers in the LAMP reactions for CDT detection were determined using two different methods, a real-time turbidity monitor and visual detection after the addition of calcein to the reaction tube. The results revealed that target DNA was amplified and visualized by these two detection methods within 60 min at a temperature of 60°C. The sensitivity of the LAMP assay was identified to be 10-fold greater than that of polymerase chain reaction analysis. When 25 alternative bacterial strains lacking CDT were tested, the results of the amplification were negative, confirming the specificity of the primers. In conclusion, the visual LAMP method established in the present study may be a rapid, reliable and cost-effective tool for detecting CDT-producing C. difficile strains at the point of care.

| S-EPMC5704324 | biostudies-literature

Loop-mediated isothermal amplification targeting 18S ribosomal DNA for rapid detection of Acanthamoeba.

Project description:Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.

| S-EPMC3712100 | biostudies-literature

Detection of canine distemper virus in blood samples by reverse transcription loop-mediated isothermal amplification.

Project description:Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was used to detect canine distemper virus (CDV) genomic RNA. A set of four primers, two outer and two inner, were designed from CDV genomic RNA targeting the nucleocapsid protein gene. The optimal reaction time and temperature for LAMP were determined to be 60 min at 65 degrees C. The relative sensitivity and specificity of RT-LAMP was found to be 100% and 93.3%, respectively, based on 50 canine blood samples and using RT-PCR as the gold standard. The detection limit of the RT-LAMP method was 100 times lower than with RT-PCR (10-1TCID50 ml(-1) versus 10TCID50 ml(-1)). In addition to the advantage resulting from the visual detection of the end-product, the LAMP method is fast, requiring only 1 h to complete the assay. The LAMP method is a viable alternative to RT-PCR for diagnosing CDV infection in dogs. The LAMP method might be useful as an on site diagnostic assay for detecting CDV.

| S-EPMC7165947 | biostudies-literature

Loop-mediated isothermal amplification method targeting the lytA gene for detection of Streptococcus pneumoniae.

Project description:It is difficult to separate Streptococcus pneumoniae from the genotypically similar species Streptococcus mitis and Streptococcus oralis, which are commensals of the human oral cavity. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63 degrees C) with high specificity, efficiency, and rapidity, was examined regarding its applicability for detecting S. pneumoniae. An S. pneumoniae-specific LAMP primer targeting the lytA gene was designed. The primer specificity was validated using 10 Streptococcus and 7 non-Streptococcus species. Within 60 min, the assay could detect 10 or more copies of purified S. pneumoniae DNA with a sensitivity 1,000 times that of conventional PCR. Clinical isolates of 21 other strains (3 S. oralis, 17 S. mitis, and 1 Streptococcus species) that harbor virulence-factor-encoding genes (lytA or ply) were tried to differentiate S. pneumoniae. The detection of S. pneumoniae in clinical isolates was more selective using the LAMP method than using conventional PCR. Therefore, LAMP appears to be a sensitive and reliable means of diagnosing S. pneumoniae infection.

| S-EPMC1081357 | biostudies-literature

Development of Rapid and Specific Detection for the Human <i>Aichivirus</i> A Using the Loop-Mediated Isothermal Amplification from Water Samples.

Project description:Human Aichivirus A (AiV-A) is classified as a Kobuvirus, group IV positive sense single strand RNA viruses. The first outbreak of AiV-A was reported from Aichi Prefecture, Japan in 1989. AiV-A exists not only among clinical patients, such as diarrhea, but also in a variety of water environments, as its occurrence is reported across a wide geographical range, from developing to advanced countries. For diagnose of AiV-A from water samples, mostly polymerase chain reaction (PCR) system have been developed. However, loop-mediated isothermal amplification (LAMP) assay has not been applied. In this study, developed a LAMP method to achieve a rapid, specific and highly sensitive detection of AiV-A. The method developed in this study is aimed specifically at AiV-A. Through a specific and non-specific selection and sensitivity test process for the five prepared LAMP primer sets, one primer set and optimum reaction temperature were selected. A newly developed method was more rapid (approximately 2-8 h), specific and equivalent detection of AiV-A than with the conventional PCRs. In addition, confirm system of positive LAMP reaction was developed by using the restriction enzyme Aci I and Hae III. For evaluation and verification of developing LAMP assay, a was applied to twenty cDNA from groundwater samples. This study proved rapid and specific diagnosis of AiV-A from water samples, and it is also demanded to be applicable to other environmental, clinical and food samples.

| S-EPMC6646635 | biostudies-literature

Development and preliminary evaluation of a loop-mediated isothermal amplification procedure for sensitive detection of cryptosporidium oocysts in fecal and water samples.

Project description:A loop-mediated isothermal amplification (LAMP) procedure for the detection of Cryptosporidium in environmental and fecal samples was developed and evaluated. This is the first demonstration of LAMP applied to detection of Cryptosporidium. Due to its specificity and simplicity, the method could become a useful diagnostic tool for epidemiologic studies of Cryptosporidium presence.

| S-EPMC2042060 | biostudies-literature

Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae using a loop-mediated isothermal amplification.

Project description:BackgroundVibrio cholerae is widely acknowledged as one of the most important waterborne pathogen causing gastrointestinal disorders. Cholera toxin (CT) is a major virulence determinant of V. cholerae. Detection of CT-producing V. cholerae using conventional culture-, biochemical- and immunological-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the sensitive and rapid detection of cholera toxin (CT)-producing Vibrio cholerae.ResultsThe assay provided markedly more sensitive and rapid detection of CT-producing V. cholerae strains than conventional biochemical and PCR assays. The assay correctly identified 34 CT-producing V. cholerae strains, but did not detect 13 CT non-producing V. cholerae and 53 non-V. cholerae strains. Sensitivity of the LAMP assay for direct detection of CT-producing V. cholerae in spiked human feces was 7.8 x 102 CFU per g (1.4 CFU per reaction). The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay for detection of CT-producing V. cholerae required less than 35 min with a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 70 min with human feces from the beginning of DNA extraction to final determination.ConclusionThe LAMP assay is a sensitive, rapid and simple tool for the detection of CT-producing V. cholerae and will be useful in facilitating the early diagnosis of human V. cholerae infection.

| S-EPMC2446398 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data