scholarly journals Point of care colourimetric and lateral flow LAMP assay for the detection of Haemonchus contortus in ruminant faecal samples

Parasite ◽  
2021 ◽  
Vol 28 ◽  
pp. 82
Author(s):  
Rojesh Khangembam ◽  
Mariann Tóth ◽  
Nóra Vass ◽  
Marián Várady ◽  
Levente Czeglédi ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Haemonchus Contortus ◽  
Extraction Method ◽  
Point Of Care ◽  
Lamp Assay ◽  
Lateral Flow ◽  
Molecular Diagnostic ◽  
Diagnostic Assay ◽  
Faecal Samples ◽  
True Point

In this study, we present an optimised colourimetric and a lateral flow LAMP assay for the detection of Haemonchus contortus in small ruminant faecal samples. Using a previously published LAMP primer set, we made use of commercially available colourimetric LAMP and lateral flow kits and combined this into an optimised diagnostic assay which was then tested on field faecal samples from Eastern and South-Eastern Hungary as well as a pure H. contortus egg faecal sample from Košice, Slovakia. Both assays showed no conflicts in visual detection of the results. Additionally, we modified and tested several centrifuge-free DNA extraction methods and one bead-beating egg lysis DNA extraction method to develop a true point of care protocol, as the source of the starting DNA is the main rate-limiting step in farm-level molecular diagnosis. Out of the various methods trialed, promising results were obtained with the magnetic bead extraction method. Sample solutions from the Fill-FLOTAC® technique were also utilised, which demonstrated that it could be efficiently adapted for field-level egg concentration to extract DNA. This proof of concept study showed that isothermal amplification technologies with a colourimetric detection or when combined with a lateral flow assay could be an important step for a true point of care molecular diagnostic assay for H. contortus.

scholarly journals Application of Loop-Mediated Isothermal Amplification combined with colorimetric and lateral flow dipstick visualization as the potential point-of-care testing for diphtheria

2019 ◽  
Author(s):  
Aleksandra Anna Zasada ◽  
Aldona Wiatrzyk ◽  
Urszula Czajka ◽  
Klaudia Brodzik ◽  
Kamila Formińska ◽  
...  
Keyword(s):  
Detection Limit ◽  
Sensitivity And Specificity ◽  
Point Of Care ◽  
Lamp Assay ◽  
Corynebacterium Diphtheriae ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Suspected Case ◽  
Point Of Care Testing ◽  
Loop Mediated Isothermal Amplification

Abstract Background Diphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA. Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts. The aim of the study was to establish simple and rapid testing methods based on Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Corynebacterium diphtheriae and differentiation between toxigenic and non-toxigenic strains.Methods Corynebacterium diphtheriae and Corynebacterium ulcerans isolates from the National Institute of Public Health-National Institute of Hygiene collection were used for the development of LAMP assay for the diagnosis of diphtheria and nontoxigenic C. diphtheriae infections. Various colorimetric methods for visualization of results were investigated. Sensitivity and specificity of the assay were examined using a collection of DNA samples from various gram-positive and gram-negative bacteria.Results The LAMP assay for tox and dtxR genes was developed. The sensitivity and specificity of the assay were calculated as 100%. The detection limit was estimated as 1.42 pg/µl concentration of DNA template when the reaction was conducted for 60 min. However, the detection limit was lowered 10 times for every 10 minutes of reduction in the time of incubation during the reaction. Positive results were successfully detected colorimetrically using hydroxynaphthol blue, calcein, QuantiFluor, and lateral flow Milenia HybriDetect dipsticks.Conclusion The assay developed in the study might be applied for point-of-care testing of diphtheria and other C. diphtheriae infections. It is highly sensitive, specific, inexpensive, easy to use, and suitable for low-resource settings.

scholarly journals Evaluation of different DNA extraction methods and loop-mediated isothermal amplification primers for the detection of Mycobacterium ulcerans in clinical specimens

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Anthony Ablordey ◽  
Evans Ahotor ◽  
Charles A. Narh ◽  
Sandra A. King ◽  
Isra Cruz ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Point Of Care ◽  
Buruli Ulcer ◽  
Extraction Methods ◽  
Isothermal Amplification ◽  
Mycobacterium Ulcerans ◽  
Loop Mediated Isothermal Amplification ◽  
Dna Extraction Method ◽  
Dna Extraction Methods

Abstract Background Early diagnosis and treatment of Buruli ulcer is critical in order to avoid the debilitating effects of the disease. In this regard, the development of new diagnostic and point of care tools is encouraged. The loop-mediated isothermal amplification for the detection of Mycobacterium ulcerans represents one of the new tools with a good potential of being developed into a point of care test. There is however the need to standardize the assays, reduce sample preparation times, improve the detection/visualization system and optimize them for high-throughput screening, adaptable to low resourced laboratories. Methods In this study, we assessed two DNA extraction protocols (modified Boom and EasyNAT methods), three previously published LAMP primer sets (BURULI, MU 2404 and BU-LAMP), and compared the sensitivity and specificity of LAMP assays on three DNA amplification platforms. Results Our results show that Buruli ulcer diagnosis using primers targeting IS2404 for the LAMP method is sensitive (73.75–91.49%), depending on the DNA extraction method used. Even though the modified Boom DNA extraction method provided the best results, its instrumentation requirement prevent it from being field applicable. The EasyNAT method on the other hand is simpler and may represent the best method for DNA extraction in less resourced settings. Conclusions For further work on the development and use of LAMP tests for Buruli diagnosis, it is recommended that the BURULI sets of primers be used, as these yielded the best results in terms of sensitivity (87.50–91.49%) and specificity (89.23–100%), depending on the DNA extraction methods used.

scholarly journals Urine-Based Molecular Diagnostic Tests for Leishmaniasis Infection in Human and Canine Populations: A Meta-Analysis

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 269
Author(s):  
Styliani A. Pappa ◽  
Panagiota I. Kontou ◽  
Pantelis G. Bagos ◽  
Georgia G. Braliou
Keyword(s):  
Dna Extraction ◽  
Diagnostic Tests ◽  
Extraction Method ◽  
Meta Analysis ◽  
Middle Eastern ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Leishmania Infection ◽  
Dna Extraction Method ◽  
The Impact

Leishmaniasis is a neglected tropical disease affecting humans and domesticated animals with high mortality in endemic countries. The pleiotropy of symptoms and the complicated gold-standard methods make the need for non-invasive, highly sensitive diagnostic tests imperative. Individual studies on molecular-based Leishmania diagnosis in urine show high discrepancy; thus, a data-evidenced comparison of various techniques is necessary. We performed a systematic review and meta-analysis using the bivariate method of diagnostic methods to pool sensitivities and specificities. We investigated the impact of DNA-extraction method, PCR type, amplified locus, host species, leishmaniasis form, and geographical region. The pooled sensitivity was 69.2%. Tests performed with the kit-based DNA extraction method and qPCR outweighed in sensitivity the phenol-chloroform-based and PCR methods, while their combination showed a sensitivity of 79.3%. Amplified locus, human or canine as host and cutaneous or visceral leishmaniasis revealed similar sensitivities. Tests in European and Middle Eastern countries performed better than tests in other regions (sensitivity 81.7% vs. 43.7%). A combination of kit-based DNA extraction and qPCR could be a safer choice for molecular diagnosis for Leishmania infection in urine samples in European–Middle Eastern countries. For the rest of the world, more studies are needed to better characterize the endemic parasite species.

scholarly journals Comparative Diagnostic Performance of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Kit for the Rapid Detection of SARS-CoV-2

Pathogens ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1629
Author(s):  
Alexander Domnich ◽  
Andrea Orsi ◽  
Donatella Panatto ◽  
Vanessa De Pace ◽  
Valentina Ricucci ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Diagnostic Performance ◽  
Point Of Care ◽  
Lamp Assay ◽  
Laboratory Diagnosis ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Rt Pcr ◽  
Laboratory Equipment ◽  
Loop Mediated Isothermal Amplification

Although the reverse transcription-polymerase chain reaction (RT-PCR) is considered a standard-of-care assay for the laboratory diagnosis of SARS-CoV-2, several limitations of this method have been described. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an alternative molecular assay and is potentially able to overcome some intrinsic shortcomings of RT-PCR. In this study, we evaluated the diagnostic performance of the novel HG COVID-19 RT-LAMP assay. In this retrospective analysis, a total of 400 routinely collected leftover nasopharyngeal samples with a known RT-PCR result were tested by means of the HG COVID-19 RT-LAMP assay. The overall sensitivity and specificity values of HG COVID-19 RT-LAMP versus RT-PCR were 97.0% (95% CI: 93.6–98.9%) and 98.5% (95% CI: 95.7–99.7%), respectively. Inter-assay agreement was almost perfect (κ = 0.96). Concordance was perfect in samples with high viral loads (cycle threshold < 30). The average time to a positive result on RT-LAMP was 17 min. HG COVID-19 RT-LAMP is a reliable molecular diagnostic kit for detecting SARS-CoV-2, and its performance is comparable to that of RT-PCR. Shorter turnaround times and the possibility of performing molecular diagnostics in the point-of-care setting make it a valuable option for facilities without sophisticated laboratory equipment.

scholarly journals Application of Loop-Mediated Isothermal Amplification combined with colorimetric and lateral flow dipstick visualization as the potential point-of-care testing for Corynebacterium diphtheriae

2020 ◽  
Author(s):  
Aleksandra Anna Zasada ◽  
Aldona Wiatrzyk ◽  
Urszula Czajka ◽  
Klaudia Brodzik ◽  
Kamila Formińska ◽  
...  
Keyword(s):  
Detection Limit ◽  
Sensitivity And Specificity ◽  
Point Of Care ◽  
Lamp Assay ◽  
Corynebacterium Diphtheriae ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Suspected Case ◽  
Point Of Care Testing ◽  
Loop Mediated Isothermal Amplification

Abstract Background Diphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA. Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts.The aim of the study was to establish simple and rapid testing methods based on Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Corynebacterium diphtheriae and differentiation between toxigenic and non-toxigenic strains.Methods Corynebacterium diphtheriae and Corynebacterium ulcerans isolates from the National Institute of Public Health-National Institute of Hygiene collection were used for the development of LAMP assay for the diagnosis of diphtheria and nontoxigenic C. diphtheriae infections. Various colorimetric methods for visualization of results were investigated. Sensitivity and specificity of the assay were examined using a collection of DNA samples from various gram-positive and gram-negative bacteria.Results The LAMP assay for tox and dtxR genes was developed. The sensitivity and specificity of the assay were calculated as 100%. The detection limit was estimated as 1.42 pg/µl concentration of DNA template when the reaction was conducted for 60 min. However, the detection limit was lowered 10 times for every 10 minutes of reduction in the time of incubation during the reaction. Positive results were successfully detected colorimetrically using hydroxynaphthol blue, calcein, QuantiFluor, and lateral flow Milenia HybriDetect dipsticks.Conclusion The assay developed in the study might be applied for point-of-care testing of diphtheria and other C. diphtheriae infections as well as for other infections caused by diphtheria-toxin producing Corynebacterium species. It is highly sensitive, specific, inexpensive, easy to use, and suitable for low-resource settings.

scholarly journals Development and evaluation of a rapid and simple diagnostic assay for COVID-19 based on loop-mediated isothermal amplification

2020 ◽  
Vol 14 (11) ◽  
pp. e0008855
Author(s):  
Rokusuke Yoshikawa ◽  
Haruka Abe ◽  
Yui Igasaki ◽  
Saeki Negishi ◽  
Hiroaki Goto ◽  
...  
Keyword(s):  
Clinical Evaluation ◽  
Rural Areas ◽  
Point Of Care ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Diagnostic Assay ◽  
Loop Mediated Isothermal Amplification ◽  
Efficient Detection ◽  
Novel Coronavirus

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic novel coronavirus that has caused a worldwide outbreak. Here we describe a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that uses a portable device for efficient detection of SARS-CoV-2. This RT-LAMP assay specifically detected SARS-CoV-2 without cross-reacting with the most closely related human coronavirus, SARS-CoV. Clinical evaluation of nasal swab samples from suspected SARS-CoV-2 pneumonia (COVID-19) patients showed that the assay could detect over 23.7 copies within 15 min with a 100% probability. Since the RT-LAMP assay can be performed with a portable battery-supported device, it is a rapid, simple, and sensitive diagnostic assay for COVID-19 that can be available at point-of-care. We also developed the RT-LAMP assay without the RNA extraction step–Direct RT-LAMP, which could detect more than 1.43 x 103 copies within 15 min with a 100% probability in clinical evaluation test. Although the Direct RT-LAMP assay was less sensitive than the standard RT-LAMP, the Direct RT-LAMP assay can be available as the rapid first screening of COVID-19 in poorly equipped areas, such as rural areas in developing countries.

Electroflotation of Escherichia coli Improves Detection Rates by Loop-Mediated Isothermal Amplification

2018 ◽  
Vol 61 (4) ◽  
pp. 1209-1220
Author(s):  
Lena Michelle Diaz ◽  
Daniel Jenkins ◽  
Ryo Kubota ◽  
Natalie Walter ◽  
Yong Li ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Pathogen Detection ◽  
Direct Detection ◽  
Point Of Care ◽  
Lamp Assay ◽  
Molecular Diagnostic ◽  
Process Conditions ◽  
Diagnostic Systems ◽  
Detection Rates ◽  
Glycerate Kinase

Abstract. The power of portable molecular diagnostic systems for detection of pathogenic microorganisms in food and environmental samples is largely limited by small assay volumes (typically 1 to 5 µL), making direct detection of trace contamination (i.e., &lt;104 CFU mL-1) unreliable. To improve detection limits for pathogens dispersed on an ecological scale, we developed a portable point-of-care (POC) sample preparation system using electroflotation (EF) to recover small quantities of these organisms from samples of hundreds of milliliters. Electrolysis reactions, supported on platinum-coated titanium electrodes, generate hydrogen and oxygen microbubbles that impel and displace suspended cells into a recovered concentrate. Samples were prepared by inoculating 380 mL of sterilized phosphate buffer (0.1 M, pH 6.6) with stock culture of ATCC 25922 to final concentrations ranging from 102 to 104 CFU mL-1. Samples were subjected to 10, 15, and 20 min durations of EF treatment under high and low turbulence conditions. We used a loop-mediated amplification (LAMP) assay with primers targeting a single-copy gene (glycerate kinase) in generic to evaluate the effects of EF treatment on concentration and recovery of detectable cell material. LAMP failed to detect in all untreated (control) samples at concentrations below 104 CFU mL-1 but was able to detect in 102 CFU mL-1 samples subjected to various conditions of EF treatment. Two-way ANOVA showed significant differences in detection rates between EF treatment durations for both high (p = 0.0019) and low turbulence (p = 0.002). Dunnett’s multiple comparison tests identified five process conditions resulting in significant (p &lt; 0.05) differences in detection between treatments and the control. Keywords: Biotechnology, Electrolysis, Food pathogens, Microbubbles, Molecular diagnostics, Pathogen detection, POC sample preparation.

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