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

Autor:	Choopara I; Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand., Teethaisong Y; Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand., Arunrut N; Bioengineering and Sensing Technology Research Team, National Center for Genetic Engineering and Biotechnology, Pathum Thani, Thailand., Thunyaharn S; Faculty of Medical Technology, Nakhonratchasima College, Nakhon Ratchasima, Thailand., Kiatpathomchai W; Bioengineering and Sensing Technology Research Team, National Center for Genetic Engineering and Biotechnology, Pathum Thani, Thailand., Somboonna N; Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.; Microbiome Research Unit for Probiotics in Food and Cosmetics, Chulalongkorn University, Bangkok, Thailand.
Jazyk:	angličtina
Zdroj:	PeerJ [PeerJ] 2021 Mar 18; Vol. 9, pp. e11082. Date of Electronic Publication: 2021 Mar 18 (Print Publication: 2021).
DOI:	10.7717/peerj.11082
Abstrakt:	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. Competing Interests: Naraporn Somboonna and Ilada Choopara hold the Thailand petty patent (detection methods for total fungi using color loop-mediated isothermal amplification, no. 1803002277, 2 Oct. 2018). (©2021 Choopara et al.)
Databáze:	MEDLINE
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