Specific and sensitive, ready-to-use universal fungi detection by visual color using ITS1 loop-mediated isothermal amplification combined hydroxynaphthol blue
| Autor: | Naraporn Somboonna, Yothin Teethaisong, Sudaluck Thunyaharn, Ilada Choopara, Wansika Kiatpathomchai, Narong Arunrut |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Loop-mediated isothermal amplification (LAMP)
Ready-to-use Loop-mediated isothermal amplification lcsh:Medicine Mycology Microbiology General Biochemistry Genetics and Molecular Biology 03 medical and health sciences chemistry.chemical_compound 18s rdna Diagnostic 030304 developmental biology Zygomycota 0303 health sciences biology Ascomycota 030306 microbiology Phylum Hydroxynaphthol blue (HNB) General Neuroscience Internal transcribed spacer (ITS) lcsh:R Fungi Basidiomycota General Medicine biology.organism_classification Hydroxynaphthol blue chemistry Ready to use General Agricultural and Biological Sciences Biotechnology |
| Zdroj: | PeerJ PeerJ, Vol 9, p e11082 (2021) |
| ISSN: | 2167-8359 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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