Comparison of Direct Sequencing with Real-time PCR High Resolution Melt and PCR Restriction Fragment Length Polymorphism Analysis to Identify Clinically Important Candida Species

Abstract

Background: Candida albicans is the predominant yeast reported from human infection. Non-albicans Candida species have been recently developed as medically vital fungi. Therefore, it is essential to detect and identify the pathogens at the species level to prescribe appropriate treatment. Methods: This study assessed two complementary methods, including real-time polymerase chain reaction-high resolution melt (PCR-HRM) and polymerase chain reaction-restriction fragment length morphism (PCR-RFLP) with standard PCR and Sanger sequencing as the benchmark. Results: In total, 66 samples were tested, and two newly-advanced assays were more effective and displayed comprehensive concordance (66/66, 100%) with Sanger sequencing outcomes. Moreover, accurate and economical tests were positively advanced by real-time PCR-HRM for C. albicans and C. parapsilosis complexes. Conclusions: Given the number of studies performed on the comparison of sensitivity and specificity of phenotypic and genotypic methods to diagnose and identify invasive fungal pathogens and the findings of this study, it could be stated that the correlative PCR-HRM and PCR-RFLP methods were effectively advanced as substitutes for conventional Sanger sequencing for the reasonable identification. However, supplementary evaluations and confirming studies should be carried out with a broad range of samples to standardize this method for routine application in medical laboratories.