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Establishment and optimization of a system for the detection of Candida albicans based on enzymatic recombinase
Xiaotong Zeng1,2, Qiuyang Jiang1,2, Fo Yang1,2
1Anhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei, Anhui, China.
Abstract:
Invasive candidiasis is a fungal infection caused by various pathogenic yeasts, with Candida albicans as the predominant pathogen. Traditional culturing and identification methods for C. albicans are slow, requiring several days to weeks to produce results, which hampers rapid diagnosis. In this study, we proposed three amplification methods to combine with CRISPR/Cas12a and selected the enzymatic recombinase amplification (ERA) and CRISPR/Cas12a two-step method for the detection of C. albicans in terms of sensitivity, and then the two-step method was optimized to a temperature-controlled one-step method for the detection of C. albicans by enzymatic recombinase amplification (ERA)-CRISPR/Cas12a. The temperature-controlled system employs a combination of liquid and solid paraffin wax to maintain the desired melting point, thus facilitating spatial separation of the ERA amplification system from the CRISPR/Cas12a detection system within a single tube. After a reaction at 37°C, the temperature is raised to 45°C, melting the wax and allowing the amplification system to merge with the detection system, initiating the reaction. This one-step detection platform simplifies and expedites the procedure, achieving a sensitivity level on par with that of two-step methods. The reaction completes in about 30 minutes, detecting as little as 100 ag/µL of genomic DNA from C. albicans pure cultures. It shows high specificity and resistance to clinical nucleic acid interference, without cross-reactivity. Additionally, the method eliminates the need to open the reaction tube, effectively preventing aerosol contamination and providing a stable, thus offering a new tool for the rapid clinical diagnosis of C. albicans.
Importance:
This study established a two-step method through optimization, compared its sensitivity, and then combined the specific detection capabilities of ERA and CRISPR/Cas12a. Furthermore, a one-step method was developed based on the two-step method, creating a one-step system for the detection of Candida albicans. This system does not require the lid to be opened during the reaction process, reducing aerosol contamination and minimizing the risk of false positives. This method does not require advanced instruments or equipment and shows strong specificity without being affected by other pathogens. It can serve as a new method for the detection of Candida albicans and has significant practical application prospects.
Insights
A new one-step method using enzymatic recombinase amplification (ERA) and CRISPR/Cas12a rapidly detects Candida albicans. This simplified assay offers high sensitivity and specificity for quick clinical diagnosis of invasive candidiasis.
Area of Science:
- Molecular Biology
- Diagnostic Microbiology
- Biotechnology
Background:
- Invasive candidiasis, a serious fungal infection, is predominantly caused by *Candida albicans*.
- Current diagnostic methods for *C. albicans* are time-consuming, delaying critical treatment.
- Rapid and accurate detection is crucial for effective management of invasive candidiasis.
Purpose of the Study:
- To develop and optimize a rapid detection method for *Candida albicans*.
- To combine enzymatic recombinase amplification (ERA) with CRISPR/Cas12a technology.
- To establish a sensitive, specific, and user-friendly diagnostic platform.
Main Methods:
- A two-step detection method using ERA and CRISPR/Cas12a was initially developed and optimized.
- A temperature-controlled one-step method was engineered using paraffin wax for spatial separation and controlled mixing of reaction components.
- The one-step method was evaluated for sensitivity, specificity, and resistance to clinical sample interference.
Main Results:
- The optimized one-step ERA-CRISPR/Cas12a method achieved sensitivity comparable to two-step approaches.
- Detection was completed within approximately 30 minutes, with a limit of detection as low as 100 ag/µL of *C. albicans* genomic DNA.
- The method demonstrated high specificity, no cross-reactivity, and resistance to nucleic acid interference in clinical samples.
Conclusions:
- The developed one-step temperature-controlled system offers a simplified and expedited procedure for *C. albicans* detection.
- This method eliminates the need to open reaction tubes, significantly reducing aerosol contamination and the risk of false positives.
- The platform requires no specialized equipment, presenting a valuable new tool for rapid clinical diagnosis of invasive candidiasis.
