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Updated: Feb 24, 2026
Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
Single nucleotide polymorphism-based multiplex PCR for identification and genotyping of the oomycete Pythium
Thidarat Rujirawat1, Thanawat Sridapan2, Tassanee Lohnoo3
1Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; Research Center, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; Molecular Medicine Program, Multidisciplinary Unit, Faculty of Science, Mahidol University, Bangkok, Thailand.
Abstract:
Pythium insidiosum causes a life-threatening infectious disease, called pythiosis, in humans and animals worldwide. Diagnosis of pythiosis is difficult and often delayed. Surgical removal of infected tissue is the main treatment option. Disabilities and death are common outcomes for pythiosis patients. Reports of Py. insidiosum infections are rising. While it would be useful for clinical, epidemiological, and microbiological studies, information on genetic variation in Py. insidiosum strains is limited. This limitation is, at least in part, due to the cost and time-requirements of DNA sequencing procedures. rDNA-sequence-based phylogenetic analyses categorize Py. insidiosum into three groups, in relation to geographic distribution: Clade-I (American strains), Clade-II (American, Asian, and Australian strains), and Clade-III (Thai and American strains). In rDNA sequence analyses, we observed single nucleotide polymorphisms (SNP) that were associated with the phylogenetic clades of Py. insidiosum. In this study, we aim to develop a multiplex PCR assay, targeting the identified SNPs, for rapid genotyping of Py. insidiosum. We also aim to assess diagnostic efficiency of the assay for identification of Py. insidiosum. Fifty-three isolates of Py. insidiosum from humans (n=35), animals (n=14), and the environment (n=4), and 22 negative-control fungi were recruited for assay evaluation. Based on the pattern of amplicons, the multiplex PCR correctly assigned phylogenetic clades in 98% of the Py. insidiosum isolates tested. The assay exhibited 100% sensitivity and specificity for identification of Py. insidiosum. The assay successfully identified and genotyped the first proven isolate of Py. insidiosum from an animal with pythiosis in Thailand. In conclusion, the multiplex PCR provided accurate, sensitive and specific results for identifying and genotyping Py. insidiosum. Thus, this multiplex-PCR assay could be a simple, rapid, and cost-effective alternative to DNA sequencing for the identification and genotyping of Py. insidiosum.
Insights
A new multiplex PCR assay rapidly identifies and genotypes Pythium insidiosum, the cause of pythiosis. This cost-effective method improves diagnosis and genetic analysis of this dangerous pathogen.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Pythium insidiosum causes severe pythiosis in humans and animals globally.
- Current diagnosis is challenging and treatment options are limited, leading to poor outcomes.
- Limited genetic data on Py. insidiosum hinders clinical and epidemiological studies.
Purpose of the Study:
- To develop a rapid multiplex PCR assay for Py. insidiosum genotyping.
- To evaluate the diagnostic efficiency of the assay for Py. insidiosum identification.
- To provide a cost-effective alternative to DNA sequencing for strain analysis.
Main Methods:
- Developed a multiplex PCR assay targeting single nucleotide polymorphisms (SNPs) associated with Py. insidiosum clades.
- Evaluated the assay using 53 Py. insidiosum isolates and 22 control fungi.
- Assessed assay accuracy, sensitivity, and specificity for identification and genotyping.
Main Results:
- The multiplex PCR correctly assigned phylogenetic clades for 98% of Py. insidiosum isolates.
- The assay demonstrated 100% sensitivity and specificity for Py. insidiosum identification.
- Successfully genotyped the first reported Py. insidiosum isolate from an animal in Thailand.
Conclusions:
- The developed multiplex PCR assay is accurate, sensitive, and specific for Py. insidiosum identification and genotyping.
- This assay offers a simple, rapid, and cost-effective tool for studying Py. insidiosum.
- The assay can aid in clinical diagnosis, epidemiology, and microbiological research of pythiosis.
