Molecular techniques for pathogen identification and fungus detection in the environment

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.

Keywords: FISH; LAMP; macroarray; medical mycology; molecular diagnostics; molecular ecology; padlock probe; pathogenic fungi; plant pathology; rolling circle amplification.

Fig. 1.

Examples of fluorescence in situ hybridisation. A. Clavariopsis aquatica growing on aquatic leaf litter, probed with 18 rRNA-targeted MY1574 domain specific probe (from Baschien et al. 2008). B. Accessibility of Tetracladium marchalianum conidia for FISH 28SrRNA-targeted species specific probe TmarchB10 (modified from Baschien et al. 2008).

Fig. 2.

DNA macroarray hybridization results from frozen cranberry fruit sample collected from Massachusetts, USA (adapted from Robideau et al. 2008). The x-ray film is overlaid with the oligonucleotide spotting pattern on DNA array membrane for cranberry fruit rot fungi. The top left pair of spots and bottom right pair of spots are oligonucleotides which served as positive controls. This array is showing positive signal for Phyllosticta, Coleophoma, Epicoccum, Godronia, Alternaria, Pestalotia, and Pilidium.

Fig. 3.

Schematic representation of the multiplex tandem–PCR procedure illustrating specific detection and identification from a blood sample containing Candida albicans and C. glabrata. Reproduced from Lau et al. (2009) with the permission of Future Medicine.

Fig. 4.

Schematic overview of the padlock principle combined with OpenArray^®, Technology for multiplex detection of three different targets (adapted from van Doorn et al. 2007).

Fig. 5.

Schematic representation of the steps in padlock probe technology coupled with hyperbranched rolling circle amplification (H-RCA) for SNPs detection. 1. The hybridization of padlock probes (containing the complementary sequences at the 5’ and 3’ ends) to the target templates. 2. During a perfect match, the probe forms a circular molecular with the aid of DNA ligase; while in the case of mismatch, no circular molecules formed. 3. Non-hybridized template will be removed during the exonucleolysis reactions (digestion by exonucleases I and III). 4. H-RCA is performed using two pre-designed primers and Bst polymerase, but no amplification will take place in the absence of a circular molecular. 5. The accumulation of dsDNA products during isothermal rolling circle amplification of DNA minicircles is monitored in a real time PCR thermocycler with the addition of SYBR green.

Fig. 6.

Schematic representation of the mechanism of LAMP. A. General location of the LAMP primer set in relation to defined regions of the target DNA. Forward (F3) and backward (B3) outer primers and forward (FIP) and backward (BIP) inner primers are indicated. B, C. Basic principle and amplification steps in LAMP. In general new DNA strands are synthesizes from the F3 and B3 primers, and these strands are recognized by FIP and BIP to start loop-mediated autocycling amplifications. The final products are stem-loop DNAs with several inverted repeats of the target DNA, and cauliflower-like structures bearing multiple loops (modified from diagrams at <http://loopamp.eiken.co.jp/e/lamp/principle.html> © Eiken Chemical.