Abstract
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.
Highlights
Fungi are widespread and generally cause human, animal and plant diseases via opportunistic infections
loop-mediated isothermal amplification (LAMP)-hydroxynaphthol blue (HNB) Regions for 6 LAMP primer sequences were designed based on conserved regions among fungal species following ITS1 sequence alignment analysis
300 fungal species have been reported, to be able to detect all opportunistic fungal pathogens via portably rapid DNA assay as effective as standard molecular technique like polymerase chain reaction (PCR) is none available owning to difficulty in massive (>40,000) sequences analyses for 6 LAMP primers design and modern technique of LAMP
Summary
Fungi are widespread and generally cause human, animal and plant diseases via opportunistic infections. Fungal diseases affected >1 billion humans and killed >1.5 million cases annually. Over 10 million cases of fungal asthma (e.g. Aspergillus spp. and Cladosporium spp.), ∼1,000,000 cases fungal keratitis (Fusarium spp., Aspergillus spp. and Candida spp.), ∼3,000,000 cases chronic pulmonary aspergillosis (Aspergillus spp.), ∼700,000 cases invasive candidiasis (Candida spp.), and ∼223,100 cases cryptococcal meningitis complicating HIV/AIDS (human immunodeficiency virus and acquired immunodeficiency syndrome) (Cryptococcus spp.), were annually found (Hawksworth, 2001; Thomas & Kaliamurthy, 2013; Denning et al, 2014; Sloan & Parris, 2014; Denning et al, 2016; Bongomin et al, 2017; Pappas et al, 2018). The severity ranges from asymptomatic to mild, or life-threatening systemic infections (Hawksworth, 2001)
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