title: A universal microarray detection method for identification of multiple Phytophthora spp. using padlock probes.
abstract: The genus Phytophthora consists of many species that cause important diseases in ornamental, agronomic, and forest ecosystems worldwide. Molecular methods have been developed for detection and identification of one or several species of Phytophthora in single or multiplex reactions. In this article, we describe a padlock probe (PLP)-based multiplex method of detection and identification for many Phytophthora spp. simultaneously. A generic TaqMan polymerase chain reaction assay, which detects all known Phytophthora spp., is conducted first, followed by a species-specific PLP ligation. A 96-well-based microarray platform with colorimetric readout is used to detect and identify the different Phytophthora spp. PLPs are long oligonucleotides containing target complementary sequence regions at both their 5' and 3' ends which can be ligated on the target into a circular molecule. The ligation is point mutation specific; therefore, closely related sequences can be differentiated. This circular molecule can then be detected on a microarray. We developed 23 PLPs to economically important Phytophthora spp. based upon internal transcribed spacer-1 sequence differences between individual Phytophthora spp. Tests on genomic DNA of many Phytophthora isolates and DNA from environmental samples showed the specificity and utility of PLPs for Phytophthora diagnostics.
authors.affiliation: Forest Research Insitute, Department of Forest Protection, Sękocin Stary, Braci Leśnej 3, 05-090 Raszyn, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Sikora
authors.forename: Els
authors.initials: E
authors.lastname: Verstappen
authors.forename: Odette
authors.initials: O
authors.lastname: Mendes
authors.forename: Cor
authors.initials: C
authors.lastname: Schoen
authors.forename: Jean
authors.initials: J
authors.lastname: Ristaino
authors.forename: Peter
authors.initials: P
authors.lastname: Bonants
mesh_terms: D000818:Animals; D003124:Colorimetry / Q000295:instrumentation; D017931:DNA Primers / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D025202:Molecular Diagnostic Techniques; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D010838:Phytophthora / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D010935:Plant Diseases / Q000469:parasitology*; D010944:Plants / Q000469:parasitology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D021903:DNA, Ribosomal Spacer; D015345:Oligonucleotide Probes