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 Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Bruna de Oliveira Coelho ◽  
Heloisa Bruna Soligo Sanchuki ◽  
Dalila Luciola Zanette ◽  
Jeanine Marie Nardin ◽  
Hugo Manuel Paz Morales ◽  
...  
Keyword(s):  
Viral Load ◽  
Internal Control ◽  
Reverse Transcription ◽  
Color Change ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
False Negative ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

Abstract Background SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results. Methods To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm. Results First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high. Conclusion This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application.

scholarly journals Optical Temperature Control Unit and Convolutional Neural Network for Colorimetric Detection of Loop-Mediated Isothermal Amplification on a Lab-On-A-Disc Platform

Sensors ◽  
2019 ◽  
Vol 19 (14) ◽  
pp. 3207 ◽  
Author(s):  
Da Ye Seul Lim ◽  
Moo-Jung Seo ◽  
Jae Chern Yoo
Keyword(s):  
High Efficiency ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
Control Unit ◽  
Dna Amplification ◽  
Heating System ◽  
Isothermal Amplification ◽  
Physical Contact ◽  
Loop Mediated Isothermal Amplification ◽  
Polymeric Substrates

Lab-on-a-disc (LOD) has emerged as a promising candidate for a point-of-care testing (POCT) device because it can effectively integrate complex fluid manipulation steps using multiple layers of polymeric substrates. However, it is still highly challenging to design and fabricate temperature measurement and heating system in non-contact with the surface of LOD, which is a prerequisite to successful realization of DNA amplification especially with a rotatable disc. This study presents a Lab-on-a-disc (LOD)-based automatic loop-mediated isothermal amplification (LAMP) system, where a thermochromic coating (<~420 µm) was used to distantly measure the chamber’s temperature and a micro graphite film was integrated into the chamber to remotely absorb laser beam with super high efficiency. We used a deep learning network to more consistently analyze the product of LAMP than we could with the naked eye. Consequently, both temperature heating and measurement were carried out without a physical contact with the surface of LOD. The experimental results show that the proposed approach, which no previous work has attempted, was highly effective in realizing LAMP in LOD.

scholarly journals Field Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Platform for the Detection of Schistosoma japonicum Infection in Oncomelania hupemsis Snails

2018 ◽  
Author(s):  
Zhi-Qiang Qin ◽  
Jing Xu ◽  
Ting Feng ◽  
Shan Lv ◽  
Yin-Jun Qian ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Point Of Care ◽  
Infected Snail ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Field Evaluation ◽  
Isothermal Amplification ◽  
Pcr Analysis ◽  
Schistosomiasis Control ◽  
Loop Mediated Isothermal Amplification

Schistosoma infection in snails can be monitored by microscopy or indirectly by sentinel mice. As both these approaches sometimes miss infections, more sensitive tests are needed, particularly in low-level transmission settings. In this study, the loop-mediated isothermal amplification (LAMP) technique, designed to detect a specific 28S ribosomal S. japonicum gene with high sensitivity, was compared to microscopy using snail samples from 51 areas endemic for schistosomiasis in five Chinese provinces. The results were compared with those by polymerase chain reaction (PCR) adding DNA sequencing as a reference when needed. The testing of pooled snail samples showed that a dilution factor of 1/50, i.e., one infected snail plus 49 non-infected ones, would still result in a positive reaction after the recommended number of amplification cycles. Testing a total of 232 pooled samples, emanating from 4,006 snail specimens, with the LAMP assay showed a 6.5% rate of infection, while traditional microscopy found only 0.04% positive samples in the same materials. Parallel PCR analysis confirmed the diagnostic accuracy of the LAMP assay, with DNA sequencing even giving LAMP a slight lead. Microscopy and the LAMP test were carried out at local schistosomiasis-control stations demonstrating that the potential of the latter assay to serve as a point-of-care (POC) test with results available within 60&ndash;90 minutes, while the more complicated PCR test had to be carried out at the National Institute of Parasitic Diseases (NIPD) in Shanghai, China. In conclusion, LAMP was found to be clearly superior to microscopy and as good as, or better, than PCR. Application of LAMP testing would be useful for surveillance and risk prediction as it requires less time than other techniques and can be used under field conditions, which improves and accelerates schistosomiasis control.

scholarly journals Detection of Pathogen-Specific Antibodies by Loop-Mediated Isothermal Amplification

2015 ◽  
Vol 22 (4) ◽  
pp. 374-380 ◽  
Author(s):  
Ian E. Burbulis ◽  
Kumiko Yamaguchi ◽  
Olga V. Nikolskaia ◽  
Sean T. Prigge ◽  
Stefan Magez ◽  
...  
Keyword(s):  
Clinical Diagnosis ◽  
Clinical Laboratory ◽  
Point Of Care ◽  
High Sensitivity ◽  
Protozoan Parasite ◽  
Isothermal Amplification ◽  
Clinical Patient ◽  
Loop Mediated Isothermal Amplification ◽  
Technical Requirements ◽  
Sensitivity Specificity

ABSTRACTLoop-mediated isothermal amplification (LAMP) is a method for enzymatically replicating DNA that has great utility for clinical diagnosis at the point of care (POC), given its high sensitivity, specificity, speed, and technical requirements (isothermal conditions). Here, we adapted LAMP for measuring protein analytes by creating a protein-DNA fusion (referred to here as a “LAMPole”) that attaches oligonucleotides (LAMP templates) to IgG antibodies. This fusion consists of a DNA element covalently bonded to an IgG-binding polypeptide (protein L/G domain). In our platform, LAMP is expected to provide the most suitable means for amplifying LAMPoles for clinical diagnosis at the POC, while quantitative PCR is more suitable for laboratory-based quantification of antigen-specific IgG abundance. As proof of concept, we measured serological responses to a protozoan parasite by quantifying changes in solution turbidity in real time. We observed a >6-log fold difference in signal between sera from vaccinated versus control mice and in a clinical patient sample versus a control. We assert that LAMPoles will be useful for increasing the sensitivity of measuring proteins, whether it be in a clinical laboratory or in a field setting, thereby improving acute diagnosis of a variety of infections.

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.

DETECTION METHODS FOR RESULTS OF A LOOP-MEDIATED ISOTHERMAL AMPLIFICATION OF DNA

2020 ◽  
Vol 65 (1) ◽  
pp. 67-72
Author(s):  
Olga A. Petrusha ◽  
E. B. Faizuloev
Keyword(s):  
Gold Nanoparticles ◽  
Real Time ◽  
Fluorescence Detection ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
Lamp Reaction ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Advantages And Disadvantages ◽  
Point Of Care Diagnostics

The loop mediated isothermal amplification (LAMP) was developed by T. Notomi et al. in 2000. It has become one of the most promising methods for point-of-care diagnostics due to its accuracy, sensitivity and ease of execution. In this review, various methods for detecting the results of the LAMP reaction are considered; their advantages and disadvantages are revealed. Methods for detecting LAMP results can be divided into indirect and direct. Indirect methods aimed at detecting changes in the chemical composition of the reaction mixture include real-time turbidimetry, fluorescence detection with calcein, colorimetric detection with hydroxynaphthol blue, and detection using modified gold nanoparticles. Direct methods based on the detection of accumulation amplicons during the reaction include fluorimetric detection with intercalating dyes, resonance fluorescence energy transfer, enzyme immunoassay, immunochromatography, using cationic polymers and gold nanoparticles. The development in the field of point-of-care diagnostics is characterized by a pronounced tendency to miniaturization, the LAMP reaction on microchips and microfluidic devices with an electrochemical or optical detection method. The most promising for the diagnosis of infectious diseases are turbidimetry methods and the use of intercalating dyes. The development of portable domestic instruments for detecting of LAMP results based on real-time fluorescence detection or turbidimetry will contribute to the widespread introduction of the method into clinical laboratory diagnostic practice. A literature research was conducted in the Pubmed ncbi based on keywords.

scholarly journals Applying Loop-mediated Isothermal Amplification (LAMP) in the Diagnosis of Malaria, Leishmaniasis and Trypanosomiasis as Point-of-Care Tests (POCTs)

2018 ◽  
Vol 18 (16) ◽  
pp. 1358-1374 ◽  
Author(s):  
M. Auxiliadora Dea-Ayuela ◽  
Cristina Galiana-Roselló ◽  
Aikaterini Lalatsa ◽  
Dolores R. Serrano
Keyword(s):  
Developing Countries ◽  
Diagnostic Tests ◽  
Point Of Care ◽  
Genetic Material ◽  
High Sensitivity ◽  
Good Alternative ◽  
Isothermal Amplification ◽  
Vector Borne Diseases ◽  
Loop Mediated Isothermal Amplification ◽  
Therapeutic Regime

One of the main objectives of the WHO is controlling transmission of parasitic protozoa vector- borne diseases. A quick and precise diagnosis is critical in selecting the optimal therapeutic regime that avoids unnecessary treatments and the emergence of resistance. Molecular assays based on Loop- Mediated Isothermal Amplification (LAMP) techniques are a good alternative to light microscopy and antigen-based rapid diagnostic tests in developing countries, since they allow for a large amount of genetic material generated from a few copies of DNA, and use primers that lead to high sensitivity and specificity, while the amplification process can be performed in isothermal conditions without the need of sophisticated equipment to interpret the results. In this review, the main advances in the development of LAMP assays for the diagnosis of malaria, leishmaniasis and Chagas' disease are discussed as well as the feasibility of their implementation in developing countries and use as point- of-care diagnostic tests.

scholarly journals Performance and Application of Commercially Available Loop-Mediated Isothermal Amplification (LAMP) Kits in Malaria Endemic and Non-Endemic Settings

Diagnostics ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 336
Author(s):  
Ulrika Morris ◽  
Berit Aydin-Schmidt
Keyword(s):  
Point Of Care ◽  
High Sensitivity ◽  
Rapid Diagnostic Tests ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Point Of Care Test ◽  
Search Terms ◽  
Polymerase Chain ◽  
Meta Analyses

Loop-mediated isothermal amplification (LAMP) is a sensitive molecular tool suitable for use as a near point-of-care test for the diagnosis of malaria. Recent meta-analyses have detailed high sensitivity and specificity of malaria LAMP when compared to microscopy, rapid diagnostic tests, and polymerase chain reaction in both endemic and non-endemic settings. Despite this, the use of malaria LAMP has primarily been limited to research settings to date. In this review, we aim to assess to what extent commercially available malaria LAMP kits have been applied in different settings, and to identify possible obstacles that may have hindered their use from being adopted further. In order to address this, we conducted a literature search in PubMed.gov using the search terms (((LAMP) OR (Loop-mediated isothermal amplification)) AND ((Malaria) OR (Plasmodium))). Focusing primarily on studies employing one of the commercially available kits, we then selected three key areas of LAMP application for further review: the performance and application of LAMP in malaria endemic settings including low transmission areas; LAMP for malaria screening during pregnancy; and malaria LAMP in returning travelers in non-endemic settings.

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