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A Novel Approach Based on Real-Time PCR with High-Resolution Melting Analysis for the Simultaneous Identification of
Daniele Chieffi1, Dafne Bongiorno2, Anna Licitra1
1Institute of Sciences of Food Production, National Research Council of Italy (CNR-ISPA), 70126 Bari, Italy.
Abstract:
Staphylococcus (S.) aureus is a pathogenic bacterium able to cause several diseases in humans and animals as well as foodborne intoxications. S. argenteus, being phenotypically and genotypically related to S. aureus, is part of the so-called S. aureus complex and recently recognized as an emerging pathogen able to cause, like S. aureus, several diseases both in humans and animals, and foodborne poisoning outbreaks. However, it has been reported that the widely used conventional PCR of Brakstad et al. [Journal of Clinical Microbiology, 30(7), 1654-1660, (1992)] targeting the thermostable nuclease gene may provide false-positive S. aureus, as it is able to amplify also S. argenteus. Here, we developed a novel two-step approach that, following the PCR of Brakstad et al. (1992), discriminates S. aureus from S. argenteus by a real-time PCR with high-resolution melting analysis (rt-PCR-HRM). In particular, targeting a polymorphic 137 bp region of the sodA gene, our developed rt-PCR-HRM method clearly discriminated S. aureus from S. argenteus, showing a remarkable difference in their amplification product melting temperatures (approximately 1.3 °C) as well as distinct melting curve shapes. The good sensitivity, reproducibility, user friendliness, and cost effectiveness of the developed method are advantageous attributes that will allow not only its easy employment to correctly identify misidentified isolates present in various collections of S. aureus, but also expand the still lacking knowledge on the prevalence and distribution of S. argenteus.
Insights
A new real-time PCR method accurately distinguishes Staphylococcus aureus from the emerging pathogen Staphylococcus argenteus. This approach improves bacterial identification, aiding in understanding pathogen prevalence and distribution.
Area of Science:
- Microbiology
- Molecular Biology
- Pathogen Detection
Background:
- Staphylococcus aureus is a significant human and animal pathogen.
- Staphylococcus argenteus, a related emerging pathogen, is often misidentified as S. aureus using conventional methods.
- Existing PCR methods targeting the nuclease gene can yield false positives for S. aureus due to cross-amplification with S. argenteus.
Purpose of the Study:
- To develop a novel method for accurate differentiation between S. aureus and S. argenteus.
- To overcome the limitations of conventional PCR in distinguishing these closely related species.
- To provide a tool for correct identification of bacterial isolates and epidemiological studies.
Main Methods:
- A two-step approach was developed, starting with conventional PCR.
- A subsequent real-time PCR with high-resolution melting analysis (rt-PCR-HRM) was employed.
- The rt-PCR-HRM targeted a polymorphic 137 bp region of the sodA gene.
Main Results:
- The developed rt-PCR-HRM method successfully discriminated S. aureus from S. argenteus.
- A significant difference in melting temperatures (approx. 1.3 °C) and distinct melting curve shapes were observed.
- The method demonstrated good sensitivity, reproducibility, and user-friendliness.
Conclusions:
- The novel rt-PCR-HRM assay provides accurate differentiation between S. aureus and S. argenteus.
- This method can correct misidentified isolates in existing collections.
- It will facilitate research on the prevalence and distribution of S. argenteus, an emerging pathogen.
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