scholarly journals A Single-Tube HNB-Based Loop-Mediated Isothermal Amplification for the Robust Detection of the Ostreid herpesvirus 1

2020 ◽  
Vol 21 (18) ◽  
pp. 6605
Author(s):  
Maja A. Zaczek-Moczydłowska ◽  
Letitia Mohamed-Smith ◽  
Anna Toldrà ◽  
Chantelle Hooper ◽  
Mònica Campàs ◽  
...  
Keyword(s):  
Point Of Care ◽  
Disease Outbreaks ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Economic Losses ◽  
Robust Detection ◽  
Loop Mediated Isothermal Amplification ◽  
Hydroxynaphthol Blue ◽  
Herpesvirus 1 ◽  
Ostreid Herpesvirus 1

The Ostreid herpesvirus 1 species affects shellfish, contributing significantly to high economic losses during production. To counteract the threat related to mortality, there is a need for the development of novel point-of-care testing (POCT) that can be implemented in aquaculture production to prevent disease outbreaks. In this study, a simple, rapid and specific colorimetric loop-mediated isothermal amplification (LAMP) assay has been developed for the detection of Ostreid herpesvirus1 (OsHV-1) and its variants infecting Crassostrea gigas (C. gigas). The LAMP assay has been optimized to use hydroxynaphthol blue (HNB) for visual colorimetric distinction of positive and negative templates. The effect of an additional Tte UvrD helicase enzyme used in the reaction was also evaluated with an improved reaction time of 10 min. Additionally, this study provides a robust workflow for optimization of primers for uncultured viruses using designed target plasmid when DNA availability is limited.

scholarly journals Earlyin plantadetection ofXanthomonas axonopodispv.punicaein pomegranate using enhanced loop-mediated isothermal amplification assay

2018 ◽  
Author(s):  
M.K. PrasannaKumar ◽  
P. Buela Parivallal ◽  
C. Manjunath ◽  
H.B. Mahesh ◽  
Karthik S Narayan ◽  
...  
Keyword(s):  
Post Infection ◽  
Bacterial Blight ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Economic Losses ◽  
Specific Primers ◽  
Loop Mediated Isothermal Amplification ◽  
Negative Results ◽  
Hydroxynaphthol Blue ◽  
Amplification Assay

AbstractBacterial blight in pomegranate caused byXanthomonas axonopodispv.punicae(Xap) is an increasing threat for pomegranate cultivation in India. To prevent the economic losses, it is pivotal to detect the infection in latent stages rather than in later stages. We have developed an enhanced method termed as loop-mediated isothermal amplification (LAMP) technique to evaluate for the latent detection of Xap in pomegranate using six set of specific primers. Three DNA intercalating dyes were used, such as Ethidium bromide, hydroxynaphthol blue (HNB) and SYBR Green resulted in visualising the positivity for LAMP assay. The reaction time and temperature were to be 65°C from 30 min onwards, for the dyes and its sensitivity was observed up to 10−7ng in the LAMP assay. For field applicability, LAMP assay detected Xap on 7thday post infection while the PCR amplified Xap after 11thday post infection. Finally, the specificity of LAMP assay was validated to be positive with ten Xap isolates for its accuracy and 29 non-Xap bacterial isolates showed negative results. Moreover, this method could be used as a better alternative to PCR based methods, for early detection of the pathogens.

scholarly journals Detection of Haemophilus parasuis isolates from South China by loop-mediated isothermal amplification and isolate characterisation

2012 ◽  
Vol 79 (1) ◽  
Author(s):  
Jian-min Zhang ◽  
Hai-yan Shen ◽  
Ming Liao ◽  
Tao Ren ◽  
Li-li Guo ◽  
...  
Keyword(s):  
16S Rrna ◽  
South China ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Economic Losses ◽  
Lamp Method ◽  
Haemophilus Parasuis ◽  
Loop Mediated Isothermal Amplification ◽  
Glässer’S Disease ◽  
Glasser's Disease

Haemophilus parasuis is the etiological agent of Glässer’s disease, which is characterised by fibrinous polyserositis, meningitis and polyarthritis, causing severe economic losses to the swine industry. In this study, a loop-mediated isothermal amplification (LAMP) test was developed to improve the specificity, facility and speed of diagnosis of H. parasuis isolates. The LAMP assay rapidly amplified the target gene within 50 min incubation at 63 °C in a laboratory water bath. The LAMP amplicon could be visualised directly in the reaction tubes following the addition of SYBR Green I dye. The detection limit of this LAMP method was 10 CFU/mL, which was 10 times more sensitive than the earlier 16S rRNA polymerase chain reaction (PCR) test conducted by Oliveira, Galina and Pijoan (2001), and no cross-reactivity was observed from other non-H. parasuis strains. This LAMP test was evaluated further on 187 clinical specimens from pigs suspected of being infected with H. parasuis. Forty-three were found positive by bacterial isolation of H. parasuis, as well as by the 16S rRNA PCR and LAMP tests. The 43 H. parasuis isolates were classified into 9 serovars and had 37 genetic patterns when analysed by pulsed-field gel electrophoresis (PFGE). This displayed that various H. parasuis serovars and genotypes were widely distributed in South China. Therefore, the speed, specificity and sensitivity of the LAMP test, the lack of a need for expensive equipment, and the visual readout showed great potential for a correct clinical diagnosis of H. parasuis in favour of controlling Glässer’s disease.

scholarly journals Specific and sensitive, ready-to-use universal fungi detection by visual color using ITS1 loop-mediated isothermal amplification combined hydroxynaphthol blue

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11082
Author(s):  
Ilada Choopara ◽  
Yothin Teethaisong ◽  
Narong Arunrut ◽  
Sudaluck Thunyaharn ◽  
Wansika Kiatpathomchai ◽  
...  
Keyword(s):  
Point Of Care ◽  
Colorimetric Detection ◽  
High Sensitivity ◽  
Fungal Species ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Fungal Isolates ◽  
Simple Detection ◽  
Hydroxynaphthol Blue ◽  
Life Threatening

Being ubiquitous, fungi are common opportunistic pathogens to humans that can lead to invasive and life-threatening infections in immunocompromised individuals. Eukaryote-resembling cell membrane and filamentous branches make the fungal diagnosis difficult. This study therefore developed a ready-to-use ITS1 loop-mediated isothermal amplification combined with hydroxynaphthol blue (LAMP-HNB) for rapid, sensitive and specific colorimetric detection of universal fungi in all phyla. The ITS1 LAMP-HNB could identify every evolutionary phylum of fungi according to sequence analyses. We tested a total of 30 clinically relevant fungal isolates (representing three major human pathogenic phyla of fungi, namely Zygomycota, Ascomycota and Basidiomycota) and 21 non-fungal isolates, and the ITS1 LAMP-HNB properly identified all isolates, with a detection limit of as low as 4.6 ag (9.6 copies), which was identical to ITS1 and 18S rDNA PCR. The assays were also validated on the feasibility of point-of-care diagnostic with real food (dry peanuts, chili and garlics) and blood samples. Furthermore, the shelf life of our ready-to-use ITS1 LAMP activity (≥50%) was more than 40 days at 30 °C with 3–5% polyvinyl alcohol or glycerol additive. The results supported the ready-to-use ITS1 LAMP-HNB for simple detection of fungi contamination with high sensitivity in local and resource-constrained areas to prevent opportunistic fungal species infections.

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 Development of a dual-gene loop-mediated isothermal amplification (LAMP) detection assay for SARS-CoV-2: A preliminary study

2020 ◽  
Author(s):  
Azeem Mehmood Butt ◽  
Shafiqa Siddique ◽  
Xiaoping An ◽  
Yigang Tong
Keyword(s):  
Point Of Care ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Nasopharyngeal Swab ◽  
Loop Mediated Isothermal Amplification ◽  
Qualitative Detection ◽  
Detection Assay ◽  
Testing Protocols ◽  
Positive Results

AbstractSevere acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as a rapidly spreading global pathogen stressing the need for development of rapid testing protocols ever than before. The aim of present study was to develop a SARS-CoV-2 detection protocol which can be performed within minimal resources and timeframe. For this purpose, we implemented the reverse transcription loop-mediated isothermal amplification (RT-LAMP) methodology for the qualitative detection of SARS-CoV-2 RNA. In order to improve the detection capability, the RT-LAMP assay was developed to simultaneously amplify two viral genes: ORF1a and N. A total of 45 SARS-CoV-2 associated coronavirus disease 2019 (COVID-19) and 25 non-COVID-19 cases were enrolled. Viral RNA was extracted from the nasopharyngeal swab samples and analyzed simultaneously using PCR and RT-LAMP protocols. Overall, our SARS-CoV-2 dual gene RT-LAMP assay was found to be 95% accurate in detecting positive cases and showed no cross-reactivity or false-positive results in non-COVID-19 samples. Further evaluation on larger and multi-centric cohorts is currently underway to establish the diagnostic accuracy and subsequent implementation into clinical practice and at point-of-care settings.

scholarly journals Comparison of a TaqMan real-time polymerase chain reaction assay with a loop-mediated isothermal amplification assay for detection of Gallid herpesvirus 1

2011 ◽  
Vol 24 (1) ◽  
pp. 138-141 ◽  
Author(s):  
Shan-Chia Ou ◽  
Joseph J. Giambrone ◽  
Kenneth S. Macklin
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Gallid Herpesvirus ◽  
Herpesvirus 1 ◽  
Polymerase Chain

A TaqMan real-time polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) assay were developed to detect Gallid herpesvirus 1 (GaHV-1, formerly Infectious laryngotracheitis virus). The standard curve of real-time PCR was established, and the sensitivity reached 10 copies/μl. In the current study, the conversion between viral titer and GaHV-1 genomic copy number was constructed. Six primers for LAMP assay amplified target gene at 65°C within 45 min, and the detection limit was 60 copies/μl. The 6 primers were highly specific, sensitive, and reproducible for detection of GaHV-1. Although the sensitivity of LAMP was lower than that of real-time PCR, LAMP was faster, less expensive, and did not require a thermocycler. The LAMP assay would be a viable alternative assay in diagnostic laboratories that do not employ real-time PCR technology.

scholarly journals Real-time loop-mediated isothermal amplification (LAMP) of mgc2 gene of Mycoplasma gallisepticum

2017 ◽  
Vol 61 (4) ◽  
pp. 439-444 ◽  
Author(s):  
Syed Ehtisham-ul-Haque ◽  
Madiha Kiran ◽  
Usman Waheed ◽  
Muhammad Younus
Keyword(s):  
Real Time ◽  
Gene Sequence ◽  
Molecular Detection ◽  
Lamp Assay ◽  
Mycoplasma Gallisepticum ◽  
Isothermal Amplification ◽  
Economic Losses ◽  
Poultry Industry ◽  
Loop Mediated Isothermal Amplification ◽  
Time Loop

AbstractIntroduction:Mycoplasma gallisepticum is considered the most pathogenic and economically significant avian Mycoplasma spp. for the worldwide poultry industry. The aim of this study was to develop a novel and sensitive real-time loop-mediated isothermal amplification (LAMP) assay based on the amplification of its mgc2 gene sequence for its rapid molecular detection in poultry.Material and Methods: Blood samples from 300 broiler and layer chickens were screened using a rapid serum agglutination (RSA) test. A real-time LAMP reaction was conducted with seropositive swab samples at 60°C for 90 min in an ESEQuant tube scanner using 6-carboxyfluorescein as the reporting dye.Results: The sensitivity of the developed assay was 10 fg/μL of DNA. The assay was found 100% specific, showing no cross-reactivity with other avian Mycoplasma species. The proportion found of the positive samples by the real-time LAMP was 58%. In comparison, the RSA was found to detect 52% of positive cases.Conclusion: The mgc2 real-time LAMP emerged as a more sensitive and accurate method for molecular detection of M. gallisepticum than RSA. Robustness and precision give it applicability as a potential field diagnostic tool for M. gallisepticum control. The study will be beneficial in reducing economic losses that M. gallisepticum inflicts on the poultry industry. This is the first reported development of a real-time LAMP assay based on the amplification of the mgc2 gene sequence using an ESEQuant tube scanner for galline M. gallisepticum detection.

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 Development of a Multiplex Loop-Mediated Isothermal Amplification Assay for Diagnosis of Plasmodium spp., Plasmodium falciparum and Plasmodium vivax

Diagnostics ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1950
Author(s):  
Woong Sik Jang ◽  
Da Hye Lim ◽  
YoungLan Choe ◽  
Hyunseul Jee ◽  
Kyung Chul Moon ◽  
...  
Keyword(s):  
Internal Control ◽  
Point Of Care ◽  
Lamp Assay ◽  
18S Rrna Gene ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Loop Mediated Isothermal Amplification

Malaria, caused by the parasite Plasmodium and transmitted by mosquitoes, is an epidemic that mainly occurs in tropical and subtropical regions. As treatments differ across species of malarial parasites, there is a need to develop rapid diagnostic methods to differentiate malarial species. Herein, we developed a multiplex malaria Pan/Pf/Pv/actin beta loop-mediated isothermal amplification (LAMP) to diagnose Plasmodium spp., P. falciparum, and P. vivax, as well as the internal control (IC), within 40 min. The detection limits of the multiplex malaria Pan/Pf/Pv/IC LAMP were 1 × 102, 1 × 102, 1 × 102, and 1 × 103 copies/µL for four vectors, including the 18S rRNA gene (Plasmodium spp.), lactate dehydrogenase gene (P. falciparum), 16S rRNA gene (P. vivax), and human actin beta gene (IC), respectively. The performance of the LAMP assay was compared and evaluated by evaluating 208 clinical samples (118 positive and 90 negative samples) with the commercial RealStar® Malaria S&T PCR Kit 1.0. The developed multiplex malaria Pan/Pf/Pv/IC LAMP assay showed comparable sensitivity (100%) and specificity (100%) with the commercial RealStar® Malaria S&T PCR Kit 1.0 (100%). These results suggest that the multiplex malaria Pan/Pf/Pv/IC LAMP could be used as a point-of-care molecular diagnostic test for malaria.

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