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

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.

A Single-Tube HNB-Based Loop-Mediated Isothermal Amplification for the Robust Detection of the 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.

Naked Eye Detection of α0 Thalassemia --SEA Type using Hydroxynaphthol Blue Loop-mediated Isothermal Amplification

BACKGROUND: Alpha-thalassemia is a major health problem for worldwide populations due to its recent large-scale global movements to many other parts of the world. The most effective method to prevent the spread of the worst form of α-thalassemia disorder, hemoglobin Bart’s hydrops fetalis, is to identify the genotypes of an α0 thalassemia carrier and then conduct subsequent genetic counseling. AIM: This research was aimed to develop a new method for the detection of α0 thalassemia (Southeast Asia or SEA) --SEA type using hydroxynaphthol blue loop-mediated isothermal amplification (HNB-LAMP) and by visually reading results using the naked eye. METHODS: This experimental research was conducted at Rangsit University, Pathum Thani Provinces, Thailand, from 2016 to 2018. Two sets of LAMP primers were used, one set of primer was used to detect the two existing α genes and the other set used in the detection of two α genes deletion --SEA types which were designed based on the nucleotide sequence of two α genes deletion from four cases of the α0 thalassemia carrier --SEA type. HNB-LAMP was developed, and then, 77 cases from subjects were evaluated by comparing them with a conventional polymerase chain reaction (PCR). RESULTS: The newly developed HNB-LAMP shows a high diagnostic sensitivity and diagnostic specificity, 100% in correct interpretation, from 10 cases of α0 thalassemia carriers --SEA type, 29 cases of α+ thalassemia carrier -α3.7 type, 8 cases of hemoglobin constant spring/hemoglobin Pakse′ (HbCS/HbPS), and 30 standard cases. CONCLUSION: This newly developed HNB-LAMP is suitable for in the field applications for detecting the presence of α0 thalassemia --SEA type due to its high diagnostic sensitivity, high diagnostic specificity, and, most notably, the ability to be detected with the naked eye.

A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing

Global efforts to combat the Covid-19 pandemic caused by the beta coronavirus SARS-CoV-2 are currently based on RT-qPCR-based diagnostic tests. However, their high cost, moderate throughput and reliance on sophisticated equipment limit widespread implementation. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative detection method that has the potential to overcome these limitations. Here we present a rapid, robust, highly sensitive and versatile RT-LAMP based SARS-CoV-2 detection assay. Our forty-minute procedure bypasses a dedicated RNA isolation step, is insensitive to carry-over contamination, and uses a hydroxynaphthol blue (HNB)-based colorimetric readout, which allows robust SARS-CoV-2 detection from various sample types. Based on this assay we have substantially increased sensitivity and scalability by a simple nucleic acid enrichment step (bead-LAMP), established a pipette-free version for home testing (HomeDip-LAMP), and developed a version with open source enzymes that could be produced in any molecular biology setting. Our advanced, universally applicable RT-LAMP assay is a major step towards population-scale SARS-CoV-2 testing.