title: Development of a nested polymerase chain reaction assay for the detection and identification of Pythium insidiosum.
abstract: Pythium insidiosum is an important cause of cutaneous and gastrointestinal disease in horses and dogs in the southeastern United States. Culture-based diagnosis of pythiosis is rarely definitive because production and identification of reproductive structures is difficult. The purpose of this study was to develop a polymerase chain reaction (PCR)-based assay for the identification of P insidiosum. Genomic DNA was extracted from 3 clinical isolates of P insidiosum and I isolate each of Pythium graminicola and Pythium arrhenomanes. The ITS I region of the ribosomal RNA gene of each isolate was amplified and sequenced, and the resultant sequences were aligned with published sequences for Pythium aphanidermatum, P acanthicum, and P myriotylum. A pair of P insidiosum-specific primers (PI-1 and PI-2) were designed from variable regions within the ITSI region. A nested PCR assay was developed in which the 1st round amplified the ITSI region by use of universal fungal primers. Second-round amplification utilized the internal P insidiosum-specific primers PI-1 and PI-2. Specificity of the assay was tested with DNA extracted from cultures of the following: 10 clinical isolates of P insidiosum and 1 isolate each of P graminicola, P irregulare, P arrhenomanes, P myriotylum, P deliense, Basidiobolus ranarum, Conidiobolus coronatus, Aspergillus terreus, Lagenidium giganteum, and a canine-pathogenic Lagenidium species. Nested PCR produced a single 105-base pair amplicon for each of the P insidiosum isolates, but did not produce amplicons for any of the other isolates. Results of this study suggest that PCR is a useful tool for the identification of P insidiosum.
authors.affiliation: Department of Veterinary Clinical Sciences, Louisiana State University, Baton Rouge 70803-8410, USA. agrooters@vetmed.lsu.edu
authors.forename: Amy M
authors.initials: AM
authors.lastname: Grooters
authors.forename: Melaney K
authors.initials: MK
authors.lastname: Gee
mesh_terms: D000818:Animals; D001483:Base Sequence; D002415:Cats; D017931:DNA Primers; D004275:DNA, Ribosomal / Q000032:analysis / Q000737:chemistry; D004285:Dogs; D005784:Gene Amplification; D020459:Genes, rRNA; D006736:Horses; D006801:Humans; D007239:Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D008969:Molecular Sequence Data; D010802:Phylogeny; D018517:Plant Roots / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D011775:Pythium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012335:RNA, Ribosomal / Q000235:genetics; D016415:Sequence Alignment / Q000662:veterinary; D012689:Sequence Homology, Nucleic Acid
chemical_list: D017931:DNA Primers; D004275:DNA, Ribosomal; D012335:RNA, Ribosomal