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

Ilada Choopara; Yothin Teethaisong; Narong Arunrut; Sudaluck Thunyaharn; Wansika Kiatpathomchai; Naraporn Somboonna

doi:10.7717/peerj.11082

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

PeerJ. 2021 Mar 18:9:e11082. doi: 10.7717/peerj.11082. eCollection 2021.

Authors

Ilada Choopara¹, Yothin Teethaisong², Narong Arunrut³, Sudaluck Thunyaharn⁴, Wansika Kiatpathomchai³, Naraporn Somboonna^{2

5}

Affiliations

¹ Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
² Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
³ Bioengineering and Sensing Technology Research Team, National Center for Genetic Engineering and Biotechnology, Pathum Thani, Thailand.
⁴ Faculty of Medical Technology, Nakhonratchasima College, Nakhon Ratchasima, Thailand.
⁵ Microbiome Research Unit for Probiotics in Food and Cosmetics, Chulalongkorn University, Bangkok, Thailand.

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.

Keywords: Diagnostic; Fungi; Hydroxynaphthol blue (HNB); Internal transcribed spacer (ITS); Loop-mediated isothermal amplification (LAMP); Ready-to-use.

Grants and funding

This research was supported by the National Research University Project, Office of Higher Education Commission (NRU59-023-HR), and the 90th Anniversary of Chulalongkorn University, Rachadapisek Sompote Fund. Ilada Choopara received financial support from the Science Achievement Scholarship of Thailand (SAST). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.