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Rapid and ultrasensitive detection of Candida tropicalis using multiple cross-displacement amplification combined
Rui Ye1,2,3, Mao Liu1,2,3, Xujian Zhang2
1Department of Basic Clinical Laboratory Medicine, School of Clinical Laboratory Science, Guizhou Medical University, Guiyang, Guizhou, China.
Background:
Candida tropicalis (C. tropicalis), classified as a World Health Organization "critical priority" pathogen, causes life-threatening invasive infections with high mortality due to diagnostic delays. Therefore, the development of rapid fungal detection platforms is an urgent scientific and clinical priority.
Methods:
We designed multiple cross-displacement amplification (MCDA) primers to target the internal transcribed spacer 2 (ITS II) gene of C. tropicalis for specific amplification. Subsequently, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-crRNA complex bound to the amplified products, activating its trans-cleavage activity to generate a detectable fluorescent signal. Finally, clinical samples were used to validate the detection results, which were further compared with those obtained using fungal culture and multiplex polymerase chain reaction (Multiplex PCR).
Results:
Under optimized conditions, the C. tropicalis-MCDA-CRISPR/Cas12a assay was completed within approximately 53 min, with a limit of detection of 30 fg of genomic DNA per reaction. The assay showed no cross-reactivity with non-C. tropicalis pathogens. Clinical validation using 128 specimens demonstrated a sensitivity of 100.0% (95% CI: 93.4-100.0%) and a specificity of 97.3% (95% CI: 90.6-99.7%) against a composite reference standard, with near-perfect agreement (κ= 0.968).
Discussion:
The C. tropicalis-MCDA-CRISPR/Cas12a assay is an efficient, accurate, and practical diagnostic tool suitable for use in resource-limited laboratories.
Insights
A new diagnostic assay rapidly detects Candida tropicalis, a critical priority pathogen. This efficient and accurate method shows high sensitivity and specificity, making it suitable for resource-limited settings.
Area of Science:
- Molecular Biology
- Diagnostic Microbiology
- Biotechnology
Background:
- Candida tropicalis is a WHO critical priority pathogen causing life-threatening infections.
- High mortality is linked to diagnostic delays, necessitating rapid detection platforms.
Purpose of the Study:
- To develop and validate a rapid and accurate diagnostic assay for Candida tropicalis.
- To address the urgent need for timely detection in clinical settings.
Main Methods:
- Designed multiple cross-displacement amplification (MCDA) primers targeting the ITS II gene of Candida tropicalis.
- Utilized CRISPR/Cas12a-crRNA complex for trans-cleavage activity and fluorescent signal generation.
- Validated the assay using clinical samples and compared results with fungal culture and multiplex PCR.
Main Results:
- The Candida tropicalis-MCDA-CRISPR/Cas12a assay achieved results within approximately 53 minutes.
- Demonstrated a limit of detection of 30 fg genomic DNA with no cross-reactivity with other pathogens.
- Clinical validation showed 100.0% sensitivity and 97.3% specificity with near-perfect agreement (κ=0.968).
Conclusions:
- The developed assay is an efficient, accurate, and practical diagnostic tool.
- The platform is suitable for use in resource-limited laboratories for Candida tropicalis detection.
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