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Use of rolling circle amplification to rapidly identify species of Cladophialophora potentially causing human
Hossein Hamzehei1, Seyed Amir Yazdanparast, Mehrnaz Mohammad Davoudi
1Laboratory Research Center, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran.
Abstract:
The genus Cladophialophora comprises etiologic agents of disease in immunocompetent patients, ranging from mild cutaneous colonization to cerebral encephalitis, in addition to saprobic species. Due to the high degree of phenotypic similarity between closely related species of the genus, identification problems are imminent. In the present study, we described rapid and sensitive rolling circle amplification (RCA) method based on species-specific padlock probes targeted for the internal transcribed spacer regions of rDNA. ITS regions of 12 Cladophialophora species were sequenced, and subsequently, 10 specific padlock probes were designed for the detection of single nucleotide polymorphisms. The majority of circularizable padlock probes were designed based on single nucleotide polymorphisms (SNPs), while for C. bantiana, C. immunda and C. devriesii were characterized by two or more nucleotides. Individual species-specific probes correctly identified in all ten Cladophialophora species correctly by visualization on 1.2 % agarose gels used to verify specificity of probe-template binding; no cross-reactivity was observed. Simplicity, sensitivity, robustness and low costs provide RCA a distinct place among isothermal techniques for DNA diagnostics. However, restriction and specificity and sensitivity should be lowered and increased, respectively, to be useful for a wide variety of clinical applications.
Insights
This study introduces a rapid rolling circle amplification (RCA) method using species-specific padlock probes for accurate Cladophialophora fungal identification. This DNA diagnostic tool offers a sensitive and cost-effective solution for distinguishing closely related species.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Genetics
Background:
- The genus Cladophialophora includes pathogenic fungi causing diverse diseases in immunocompetent individuals.
- High phenotypic similarity among Cladophialophora species presents significant identification challenges.
- Accurate species identification is crucial for understanding disease pathogenesis and epidemiology.
Purpose of the Study:
- To develop a rapid, sensitive, and species-specific DNA-based diagnostic method for Cladophialophora species.
- To utilize rolling circle amplification (RCA) with padlock probes targeting internal transcribed spacer (ITS) regions of rDNA.
- To address the limitations of traditional identification methods due to phenotypic similarities.
Main Methods:
- Sequencing of ITS regions from 12 Cladophialophora species.
- Design of 10 species-specific padlock probes targeting single nucleotide polymorphisms (SNPs) within the ITS regions.
- Application of rolling circle amplification (RCA) for probe-template binding and signal amplification.
- Detection of amplified products via agarose gel electrophoresis to confirm species-specific binding.
Main Results:
- The developed RCA method successfully identified all 10 Cladophialophora species with high specificity.
- No cross-reactivity was observed between probes and non-target species.
- The method demonstrated simplicity, sensitivity, robustness, and cost-effectiveness.
Conclusions:
- Rolling circle amplification (RCA) with species-specific padlock probes is a viable isothermal DNA diagnostic technique for Cladophialophora identification.
- The method offers a significant advancement over traditional identification techniques for this fungal genus.
- Further optimization of specificity and sensitivity is needed for broader clinical applications.
