Frontiers in Cellular and Infection Microbiology (Nov 2024)
A novel, rapid, ultrasensitive diagnosis platform for detecting Candida albicans using restriction endonuclease‐mediated real-time loop-mediated isothermal amplification
Abstract
IntroductionCandida albicans (C. albicans, CA) is an essential invasive fungus in clinical diagnosis. Although several detection methods exist, none meet the need for early diagnosis. A rapid, sensitive, and specific diagnostic tool is crucial for effective prevention and control of C. albicans infections.MethodsThis study aimed to develop a new, rapid, and ultrasensitive diagnostic tool for C. albicans detection based on restriction endonuclease-mediated real-time loop-mediated isothermal amplification (ERT-LAMP-CA). The ERT-LAMP-CA technology combines LAMP amplification, restriction endonuclease cleavage, and real-time fluorescence detection in a single reaction tube, which can complete a diagnosis of C. albicans in a short time (approximately 1 h).ResultsHerein, we developed the primer sequences required for ERT-LAMP-CA based on the ITS2 gene of C. albicans and found that ERT-LAMP-CA limit of detection was approximately 500 ag/μL genomic DNA and can present negative results for non-C. albicans templates. We tested sputum samples from 64 patients with suspected C. albicans infections to validate ERT-LAMP-CA applicability in clinical sample testing and found that ERT-LAMP-CA was consistent with multiplex PCR-capillary electrophoresis.DiscussionIn conclusion, ERT-LAMP-CA is a rapid, accurate, and sensitive assay with excellent potential for clinical and basic laboratory diagnosis and an efficient screening strategy.
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