A high density COX1 barcode oligonucleotide array for identification and detection of species of Penicillium subgenus Penicillium

Abstract

We developed a COX1 barcode oligonucleotide array based on 358 sequences, including 58 known and two new species of Penicillium subgenus Penicillium, and 12 allied species. The array was robotically spotted at near microarray density on membranes. Species and clade-specific oligonucleotides were selected using the computer programs SigOli and Array Designer. Robotic spotting allowed 768 spots with duplicate sets of perfect match and the corresponding mismatch and positive control oligonucleotides, to be printed on 2 × 6 cm 2 nylon membranes. The array was validated with hybridizations between the array and digoxigenin (DIG)-labelled COX1 polymerase chain reaction amplicons from 70 pure DNA samples, and directly from environmental samples (cheese and plants) without culturing. DNA hybridization conditions were optimized, but undesired cross-reactions were detected frequently, reflecting the relatively high sequence similarity of the COX1 gene among Penicillium species. Approximately 60% of the perfect match oligonucleotides were rejected because of low specificity and 76 delivered useful group-specific or species-specific reactions and could be used for detecting certain species of Penicillium in environmental samples. In practice, the presence of weak signals on arrays exposed to amplicons from environmental samples, which could have represented weak detections or weak cross reactions, made interpretation difficult for over half of the oligonucleotides. DNA regions with very few single nucleotide polymorphisms or lacking insertions/deletions among closely related species are not ideal for oligonucleotidebased diagnostics, and supplementing the COX1-based array with oligonucleotides derived from additional genes would result in a more robust hierarchical identification system.

Molecular techniques for pathogen identification and fungus detection in the environment

“…2001, Zhang et al 1991). It is now considered a powerful and practical technique for the detection and identification of fungi and other microbes, such as bacteria, from complex environmental samples without the need for isolation in culture (Chen et al ., 2009, Ehrmann et al . 1994, Lévesque et al 1998, Tambong et al 2006, Uehara et al .…”

“…For this type of application, oligonucleotides, or microcodes (Summerbell et al . 2005), are designed from a taxonomically complete dataset of suitable genome region(s) (Chen et al . 2009, Tambong et al .…”

Recent Diagnostic Techniques in Mycology

“…PNA (peptide nucleic acid) probes are mimics in which the negatively charged sugar-backbone of DNA is replaced with a non-charged polyamide backbone. PNA probes penetrate cell walls more effectively due to their neutrality and they do not have to overcome the destabilizing electrostatic repulsion during hybridisation [18,19].…”

Rationale for a polyphasic approach in the identification of mycotoxigenic fungi

“…In mycology, sequence-based identification, barcoding and microcoding have been suggested for identification of common filamentous fungi, but there are still many technical, economical and bioinformative problems to be solved before this may eventually become a common practice (Summerbell et al, 2005;Seifert et al, 2007;Seifert, 2009), so for both scientific and practical reasons, polyphasic identification will probably be the way to identify fungi correctly. For example in Penicillium, a known genus with many species producing mycotoxins, COX1 and ITS gene sequences will help in identification, but are not sufficiently informative to allow correct identification of all Penicillium species (Seifert et al, 2007;Chen et al, 2009), while β-tubulin sequences are more effective, but still not entirely sufficient to allow species level identification in all cases . Identification should thus be polyphasic and an identified organism should be compared to a control, a well-known authenticated isolate from a culture collection, in order to be sure it is correctly identified.…”