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.
Publication Date: 2002-03-20 PubMed ID: 11899029DOI: 10.1892/0891-6640(2002)0162.3.co;2Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research developed an effective method for diagnosing pythiosis, a disease affecting horses and dogs, using a newly established polymerase chain reaction (PCR) test that specifically detects the Pythium insidiosum causing agent, proving to be more accurate than conventional culture-based diagnosis techniques.
Objective of Research
- The main aim of this study was to create a more accurate PCR-based test to detect and identify P. insidiosum, a prime reason for skin and stomach disease in horses and dogs, particularly in the southeastern US. Earlier diagnosis techniques, which were culture-based, often did not provide definite results due to the complex nature of producing and identifying P. insidiosum’s reproductive structures.
Method of Research
- Genomic DNA was extracted from three specifically isolated P. insidiosum cases and one case each of Pythium graminicola and Pythium arrhenomanes. They amplified and sequenced the ITS I section of the ribosomal RNA gene in each case.
- The acquired sequences were lined up with published sequences for 3 species of Pythium. From the variable sections within the ITSI region, two specific primers for P. insidiosum (PI-1 and PI-2) were developed.
- A 2-round nested PCR test was developed. The first round amplified the ITSI region using universal fungal primers. The second round employed the PI-1 and PI-2 primers.
Testing and Results
- The PCR test’s specificity was assessed using DNA extracted from 14 different cultures including various Pythium species, Basidiobolus ranarum, Conidiobolus coronatus, Aspergillus terreus, Lagenidium giganteum, and a species of Lagenidium harmful to dogs.
- The nested PCR test produced a single 105-base pair amplicon for each of the P. insidiosum cases, proving the effectiveness of the primers, but created no amplicons for any other isolates. These results suggest that the novel PCR test is a useful tool for identifying P. insidiosum.
Cite This Article
APA
Grooters AM, Gee MK.
(2002).
Development of a nested polymerase chain reaction assay for the detection and identification of Pythium insidiosum.
J Vet Intern Med, 16(2), 147-152.
https://doi.org/10.1892/0891-6640(2002)0162.3.co;2 Publication
Researcher Affiliations
- Department of Veterinary Clinical Sciences, Louisiana State University, Baton Rouge 70803-8410, USA. agrooters@vetmed.lsu.edu
MeSH Terms
- Animals
- Base Sequence
- Cats
- DNA Primers
- DNA, Ribosomal / analysis
- DNA, Ribosomal / chemistry
- Dogs
- Gene Amplification
- Genes, rRNA
- Horses
- Humans
- Infections / diagnosis
- Infections / microbiology
- Infections / veterinary
- Molecular Sequence Data
- Phylogeny
- Plant Roots / microbiology
- Polymerase Chain Reaction / methods
- Polymerase Chain Reaction / veterinary
- Pythium / classification
- Pythium / genetics
- Pythium / isolation & purification
- RNA, Ribosomal / genetics
- Sequence Alignment / veterinary
- Sequence Homology, Nucleic Acid
Citations
This article has been cited 23 times.- Sridapan T, Krajaejun T. Nucleic Acid-Based Detection of Pythium insidiosum: A Systematic Review.. J Fungi (Basel) 2022 Dec 23;9(1).
- Elshafie NO, Hanlon J, Malkawi M, Sayedahmed EE, Guptill LF, Jones-Hall YL, Santos AP. Nested PCR Detection of Pythium sp. from Formalin-Fixed, Paraffin-Embedded Canine Tissue Sections.. Vet Sci 2022 Aug 19;9(8).
- Krajaejun T, Rujirawat T, Lohnoo T, Yingyong W, Sae-Chew P, Reamtong O, Kittichotirat W, Patumcharoenpol P. Secretome Profiling by Proteogenomic Analysis Shows Species-Specific, Temperature-Dependent, and Putative Virulence Proteins of Pythium insidiosum.. J Fungi (Basel) 2022 May 20;8(5).
- Rodríguez N, Whitfield-Cargile CM, Chamoun-Emanuelli AM, Hildreth E, Jordan W, Coleman MC. Nasopharyngeal bacterial and fungal microbiota in normal horses and horses with nasopharyngeal cicatrix syndrome.. J Vet Intern Med 2021 Nov;35(6):2897-2911.
- do Carmo PMS, Uzal FA, Riet-Correa F. Diseases caused by Pythium insidiosum in sheep and goats: a review.. J Vet Diagn Invest 2021 Jan;33(1):20-24.
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