Molecular techniques for pathogen identification and fungus detection in the environment

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Abstract

Many species of fungi can cause disease in plants, animals and humans. Accurate and robust detection and quantification of fungi is essential for diagnosis, modeling and surveillance. Also direct detection of fungi enables a deeper understanding of natural microbial communities, particularly as a great many fungi are difficult or impossible to cultivate. In the last decade, effective amplification platforms, probe development and various quantitative PCR technologies have revolutionized research on fungal detection and identification. Examples of the latest technology in fungal detection and differentiation are discussed here.

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Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases

Yasuyoshi Mori, Tsugunori Notomi (2009)

Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. This technology has been developed into commercially available detection kits for a variety of pathogens including bacteria and viruses. The current focus on LAMP methodology is as a diagnostic system to be employed in resource-limited laboratories in developing countries, where many fatal tropical diseases are endemic. The combination of LAMP and novel microfluidic technologies such as Lab-on-a-chip may facilitate the realization of genetic point-of-care testing systems to be used by both developed and developing countries in the near future. This review will describe the historical, current, and future developments of such technologies.

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Mutation detection and single-molecule counting using isothermal rolling-circle amplification.

P M Lizardi, X. Huang, Z. Zhu … (1998)

Rolling-circle amplification (RCA) driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. In the presence of two primers, one hybridizing to the + strand, and the other, to the - strand of DNA, a complex pattern of DNA strand displacement ensues that generates 10(9) or more copies of each circle in 90 minutes, enabling detection of point mutations in human genomic DNA. Using a single primer, RCA generates hundreds of tandemly linked copies of a covalently closed circle in a few minutes. If matrix-associated, the DNA product remains bound at the site of synthesis, where it may be tagged, condensed and imaged as a point light source. Linear oligonucleotide probes bound covalently on a glass surface can generate RCA signals, the colour of which indicates the allele status of the target, depending on the outcome of specific, target-directed ligation events. As RCA permits millions of individual probe molecules to be counted and sorted using colour codes, it is particularly amenable for the analysis of rare somatic mutations. RCA also shows promise for the detection of padlock probes bound to single-copy genes in cytological preparations.

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Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria.

Annelie Pernthaler, Jakob Pernthaler, Rudolf Amann (2002)

Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. After concentration of samples onto membrane filters and subsequent embedding of filters in low-gelling-point agarose, no decrease in bacterial cell numbers was observed during 90 min of lysozyme incubation (10 mg ml(-1) at 37 degrees C). The detection rates of coastal North Sea bacterioplankton by CARD-FISH with a general bacterial probe (EUB338-HRP) were significantly higher (mean, 94% of total cell counts; range, 85 to 100%) than that with a monolabeled probe (EUB338-mono; mean, 48%; range, 19 to 66%). Virtually no unspecific staining was observed after CARD-FISH with an antisense EUB338-HRP. Members of the marine SAR86 clade were undetectable by FISH with a monolabeled probe; however, a substantial population was visualized by CARD-FISH (mean, 7%; range, 3 to 13%). Detection rates of EUB338-HRP in Wadden Sea sediments (mean, 81%; range, 53 to 100%) were almost twice as high as the detection rates of EUB338-mono (mean, 44%; range, 25 to 71%). The enhanced fluorescence intensities and signal-to-background ratios make CARD-FISH superior to FISH with directly labeled oligonucleotides for the staining of bacteria with low rRNA content in the marine environment.

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Author and article information

Journal

Journal ID (nlm-ta): IMA Fungus

Journal ID (iso-abbrev): IMA Fungus

Journal ID (publisher-id): IMA Fungus

Title: IMA Fungus : The Global Mycological Journal

Publisher: Nationaal Herbarium Nederland & Centraallbureau voor Schimmelcultures

ISSN (Print): 2210-6340

ISSN (Electronic): 2210-6359

Publication date (Electronic): 18 November 2011

Publication date (Print): December 2011

Volume: 2

Issue: 2

Pages: 177-189

Affiliations

[1 ]Department of Forest Sciences, The University of British Columbia, Vancouver, BC, V6T 1Z4, Canada;

[2 ]The Food and Environment Research Agency, Sand Hutton, York YO41 1LZ, UK

[3 ]Central Experimental Farm, Agriculture and Agri-Food Canada, Ottawa, Canada, K1A OC6

[4 ]Centre for Infectious Diseases and Microbiology and the University of Sydney, Westmead Hospital, Westmead, NSW 2145, Australia

[5 ]Plant Research International, Business Unit Bio-Interactions and Plant Health, PO Box 16, 6700 AA, Wageningen, The Netherlands

[6 ]Technische Universität Berlin, Environmental Microbiology, Sekr. FR1-2, Franklinstrasse 29, 10587 Berlin, Germany

[7 ]CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

[8 ]Department of Parasitology and Mycology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

*Current mailing address: Department of Laboratory Medicine, 10 Center Drive, National Institutes of Health, Bethesda, MD 20892, USA

**Current mailing address: Federal Environment Agency Germany, Corrensplatz 1, 14195 Berlin, Germany

Author notes

corresponding author e-mail: clementsui@ 123456gmail.com

Article

DOI: 10.5598/imafungus.2011.02.02.09

PMC ID: 3359816

PubMed ID: 22679603

SO-VID: 6d13541c-fdb1-4858-9028-78a07e1ffb60

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Molecular techniques for pathogen identification and fungus detection in the environment

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Abstract

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Plant MYBs

Most cited references 59

Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases

Mutation detection and single-molecule counting using isothermal rolling-circle amplification.

Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria.

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