Related Experiment Video
Updated: Jun 6, 2025
Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System
Published on: April 25, 2025
End-point RPA-CRISPR/Cas12a-based detection of Enterocytozoon bieneusi nucleic acid: rapid, sensitive and specific
Yilin Wang1,2, Fuchang Yu1,2,3, Kaihui Zhang1,2
1College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan, P. R. China.
Abstract:
Enterocytozoon bieneusi is a common species of microsporidia that infects humans and animals. Current methods for detecting E. bieneusi infections have trade-offs in sensitivity, specificity, simplicity, cost and speed and are thus unacceptable for clinical application. We tested the effectiveness of a previously reported CRISPR/Cas12a-based method (ReCTC) when used for the nucleic acid detection of E. bieneusi. The limit of detection (LOD) and the specificity of the expanded ReCTC were evaluated using prepared target DNA, and the accuracy of the ReCTC-based detection of E. bieneusi in clinical samples was validated. The ReCTC method was successfully used for the nucleic acid detection of E. bieneusi. The sensitivity test indicated an LOD of 3.7 copies/µl for the ReCTC-based fluorescence and lateral flow strip methods. In specificity test involving other common enteric pathogens, a fluorescent signal and/or test line appeared only when the sample was positive for E. bieneusi. These results demonstrated that the ReCTC method can successfully detect E. bieneusi in clinical samples. The ReCTC method was successfully used to detect E. bieneusi nucleic acid with high sensitivity and specificity. It had excellent performance in clinical DNA samples and was superior to nested polymerase chain reaction. Furthermore, the ReCTC method demonstrated its capability for use in on-site detection.
Insights
A new CRISPR/Cas12a (ReCTC) method accurately detects Enterocytozoon bieneusi, a common microsporidia parasite. This sensitive and specific nucleic acid detection offers a promising alternative for clinical diagnostics.
Area of Science:
- Microbiology
- Molecular Biology
- Parasitology
Background:
- Enterocytozoon bieneusi is a prevalent microsporidian species causing infections in humans and animals.
- Existing detection methods for E. bieneusi infections lack the required sensitivity, specificity, simplicity, cost-effectiveness, and speed for clinical use.
Purpose of the Study:
- To evaluate the effectiveness of a CRISPR/Cas12a-based nucleic acid detection method, termed ReCTC, for Enterocytozoon bieneusi.
- To assess the limit of detection (LOD), specificity, and accuracy of the ReCTC method in clinical samples.
Main Methods:
- The study utilized a CRISPR/Cas12a-based system (ReCTC) for nucleic acid detection.
- Sensitivity was determined by evaluating the limit of detection (LOD) using prepared target DNA.
- Specificity was tested against other common enteric pathogens, and accuracy was validated in clinical samples.
Main Results:
- The ReCTC method successfully detected Enterocytozoon bieneusi nucleic acid.
- The ReCTC method demonstrated a high sensitivity with an LOD of 3.7 copies/µl for both fluorescence and lateral flow strip assays.
- Specificity tests confirmed that the ReCTC method only produced a signal for E. bieneusi, showing no cross-reactivity with other enteric pathogens. The method outperformed nested PCR in clinical DNA samples.
Conclusions:
- The ReCTC method provides highly sensitive and specific nucleic acid detection of Enterocytozoon bieneusi.
- The CRISPR/Cas12a-based ReCTC assay shows excellent performance in clinical samples and is suitable for on-site detection.
- ReCTC offers a superior alternative to existing methods, including nested PCR, for diagnosing E. bieneusi infections.
