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Updated: Jun 9, 2025
Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications
Published on: November 5, 2020
A highly sensitive and specific real-time quantitative polymerase chain reaction assay for Perkinsus marinus
Seung-Hyeon Kim1, S D N K Bathige1,2, Hyoun Joong Kim1,2
1Department of Aquatic Life Medicine, College of Ocean Science and Technology, Kunsan National University, 558 Daehakro, Gunsan, 54150, South Korea.
Abstract:
Oyster aquaculture is one of the fastest-growing food production industries worldwide; however, it faces a significant challenge from the protist Perkinsus marinus, particularly in the USA. Although several quantitative molecular diagnostic methodologies are available for identifying diseases caused by P. marinus, the primer pairs used therein led to non-specific identification of other Perkinsus spp. Hence, a quantitative real-time PCR (Pm-qPCR) assay specific for P. marinus was developed using a TaqMan-based probe with the internal quencher in this study. A primer pair and probe specific to P. marinus were designed from a hypothetical protein of P. marinus collected from the whole-genome shotgun sequence database of the National Center for Biotechnology Information (NCBI). In silico analysis using homologous sequences of P. olseni and P. chesapeaki confirmed the high specificity of primers designed in this study. The Pm-qPCR assay was performed using seven different strains of P. marinus, P. olseni, and P. chesapeaki, revealing high specificity and sensitivity for detecting only P. marinus strains. In conclusion, it was demonstrated that Pm-qPCR can effectively and accurately diagnose P. marinus with high specificity and sensitivity. This assay is promising for monitoring oyster health and disease management in ecosystems and aquaculture.
Insights
A new quantitative real-time PCR (qPCR) assay specifically detects Perkinsus marinus, a parasite threatening oyster aquaculture. This highly sensitive and specific method improves disease diagnosis and oyster health management.
Area of Science:
- Marine Biology
- Aquaculture Science
- Molecular Diagnostics
Background:
- Oyster aquaculture is a rapidly expanding global industry.
- Perkinsus marinus poses a significant threat to oyster populations, especially in the USA.
- Existing diagnostic methods for P. marinus lack specificity, often cross-reacting with other Perkinsus species.
Purpose of the Study:
- To develop a highly specific quantitative real-time PCR (qPCR) assay for the accurate detection of Perkinsus marinus.
- To overcome the limitations of non-specific primer pairs in current diagnostic methodologies.
Main Methods:
- Designed a novel primer pair and TaqMan-based probe specific to P. marinus, targeting a hypothetical protein.
- Utilized in silico analysis with homologous sequences of P. olseni and P. chesapeaki to confirm primer specificity.
- Validated the developed Pm-qPCR assay using seven different strains of P. marinus, P. olseni, and P. chesapeaki.
Main Results:
- The developed Pm-qPCR assay demonstrated high specificity, accurately detecting only P. marinus strains.
- The assay exhibited high sensitivity in identifying P. marinus.
- In silico analysis confirmed the specificity of the designed primers against related species.
Conclusions:
- The novel Pm-qPCR assay provides an effective and accurate method for diagnosing P. marinus infections.
- This assay offers a promising tool for monitoring oyster health and managing P. marinus diseases in aquaculture and natural ecosystems.
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