Comparative Analysis of Real time PCR and RPA for Rapid and Sensitive Detection of Candida albicans in Invasive Candidiasis

다국어 초록 (Multilingual Abstract)

Comparative Analysis of Real time PCR and RPA for Rapid Sensitive Detection of Candida albicans in Invasive Candidiasis Kim, Ah Yeoung Department of Clinical Laboratory Science Graduate School, Catholic University of Pusan Advisor : Professor Kim, Jungho Ph.D. Candida albicans is the principal etiological agent of invasive candidiasis, a severe opportunistic fungal infection associated with high morbidity and mortality, particularly among immunocompromised individuals, transplant recipients, and critically ill patients. Due to the nonspecific clinical manifestations of invasive candidiasis and the inherent limitations of traditional culture based diagnostics such as low sensitivity and prolonged turnaround time there is a critical need for rapid, sensitive and species-specific molecular diagnostic tools capable of enabling early detection and timely therapeutic intervention. In this study, we aimed to develop and comparatively evaluate C. albicans specific primer sets optimized for two molecular diagnostic platforms: quantitative Real time PCR and recombinase polymerase amplification (RPA). Primer design was performed using conserved regions of the C. albicans 28S rRNA gene, complemented by comparative sequence analysis incorporating the 26S rRNA regions of two clinical isolates to enhance species specificity. Independent primer sets were subsequently optimized based on sequence alignment, melting temperature, GC content, structural stability, and platform-specific amplification requirements. Performance evaluation focused primarily on specificity and analytical sensitivity. Specificity testing demonstrated that both primer sets exclusively amplified C. albicans genomic DNA, with no amplification observed in non-albicans Candida species, including C. glabrata, C. tropicalis, and C. parapsilosis. To assess potential nonspecific signals from common clinical contaminants, two representative Gram-positive bacteria (Staphylococcus aureus and Staphylococcus epidermidis) and two representative Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) were also tested, and no nonspecific amplification was detected. These findings confirm that the 28S/26S rRNA based primer design strategy achieved high species discrimination. Analytical sensitivity assessment demonstrated that both Real time PCR and RPA assays consistently detected genomic DNA at levels as low as approximately 1 × 10² copies. All amplification reactions were analyzed using the CFX96 Real-Time Detection System (Bio-Rad, USA), which enabled detailed monitoring of fluorescence intensity and amplification curve progression. This allowed direct comparison of Cq values, amplification efficiency, and real-time fluorescence accumulation between Real time PCR and the rapid isothermal signal initiation characteristic of Real time RPA. Real time PCR exhibited excellent reproducibility and strong quantitative performance, whereas RPA achieved amplification within 30 minutes under isothermal conditions without requiring sophisticated instrumentation, demonstrating suitability for point-of-care testing (POCT) and resource-limited settings. Overall, this study establishes that the newly developed 28S/26S rRNA–based primer sets provide highly specific and sensitive detection of C. albicans across two distinct amplification platforms. The comparative evaluation underscores their complementary diagnostic advantages and supports the development of efficient molecular diagnostic strategies for improving early detection and clinical management of invasive candidiasis.
Key words: Candida albicans, invasive candidiasis, Real time PCR, RPA, POCT

목차 (Table of Contents)

• Abstract ⅰ
• List of Tables v
• List of Figures ⅵ
• Ⅰ. Introduction 1
• Ⅱ. Materials and Methods 7
• 1. Sample collection 7
• 2. Real time PCR and RPA primer design 8
• 3. Genomic DNA extraction 11
• 4. Real time PCR and RPA Amplification Protocols 12
• Ⅲ. Results 15
• 1. Specificity analysis of Real time PCR and RPA assays 15
• 2. Sensitivity analysis of Real time PCR and RPA assays 19
• 3. Detection performance of Real time PCR and RPA assays for Clinical isolate sample 24
• Ⅳ. Discussion 28
• References 31
• Abstract in Korean 35