Rapid and simple colorimetric detection of quiescent Colletotrichum in harvested fruit using reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) technology

Manpreet Kaur; Khadijah Ayarnah; Danielle Duanis-Assaf; Noam Alkan; Evgeni Eltzov

doi:10.1016/j.talanta.2023.124251

Rapid and simple colorimetric detection of quiescent Colletotrichum in harvested fruit using reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) technology

Talanta. 2023 Apr 1:255:124251. doi: 10.1016/j.talanta.2023.124251. Epub 2023 Jan 5.

Authors

Manpreet Kaur¹, Khadijah Ayarnah¹, Danielle Duanis-Assaf¹, Noam Alkan², Evgeni Eltzov³

Affiliations

¹ Department of Postharvest Science, Institute of Postharvest and Food Sciences, The Volcani Institute, Agricultural Research Organization, Bet Dagan, 50250, Israel; Institute of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, 76100, Israel.
² Department of Postharvest Science, Institute of Postharvest and Food Sciences, The Volcani Institute, Agricultural Research Organization, Bet Dagan, 50250, Israel.
³ Department of Postharvest Science, Institute of Postharvest and Food Sciences, The Volcani Institute, Agricultural Research Organization, Bet Dagan, 50250, Israel; Agro-Nanotechnology and Advanced Materials Research Center, Institute of Postharvest and Food Science, Department of Postharvest Science, Agricultural Research Organization, The Volcani Institute, Rishon LeZion, 7505101, Israel. Electronic address: eltzov@volcani.agri.gov.il.

PMID: 36630787
DOI: 10.1016/j.talanta.2023.124251

Abstract

Anthracnose, caused by the fungus Colletotrichum gloeosporioides, is one of the major causes of postharvest decay of fruits and vegetables. Detection of the pathogen at an early stage of infection is crucial to developing a disease management strategy. In this work, a loop-mediated isothermal amplification (LAMP) assay was developed for the rapid detection of C. gloeosporioides targeting the transcript enoyl-CoA hydratase (ECH) that significantly upregulates only during C. gloeosporioides quiescent stage. The assay enabled a naked-eye detection of C. gloeosporioides RNA within 23 min based on a color change of LAMP products from pink to yellow. The detection limit of the LAMP assay was 1 pg of total RNA extracted from fruit peel in a 25 μL reaction. Positive results were obtained only in samples carrying the ECH gene, whereas no cross-reaction was observed for a different quiescent marker (histone deacetylase (HDAC)) or an appressorium marker (scytalone dehydratase, (SD)), indicating the high specificity of the method. Hence, the results indicate that the developed LAMP assay is a rapid, highly sensitive, and specific tool for the early detection of quiescent C. gloeosporioides and could be employed to manage postharvest diseases.

Keywords: Colletotrichum; LAMP assay; Naked-eye detection; Postharvest disease; Quiescent.

MeSH terms

Colletotrichum* / genetics
Colorimetry
Fruit* / microbiology
Nucleic Acid Amplification Techniques / methods
RNA
Sensitivity and Specificity
Technology

Substances

RNA

Supplementary concepts

LAMP assay