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Published on: March 22, 2012
Development and clinical implications of a novel CRISPR-based diagnostic test for pulmonary Aspergillus fumigatus
Zhengtu Li1, Mingdie Wang1, Teng Xu2
1State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, National Center for Respiratory Medicine, Guangzhou 510120, China.
Background:
Rapid and reliable diagnostic methods for Aspergillus fumigatus infection are urgently needed. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 13a (Cas13a) has high sensitivity and specificity in the diagnosis of viral infection. However, its potential use in detecting A. fumigatus remains unexplored. A highly sensitive and specific method using the CRISPR/Cas13a system was developed for the reliable and rapid detection of A. fumigatus.
Methods:
The conserved internal transcribed spacer (ITS) region of A. fumigatus was used to design CRISPR-derived RNA (crRNA) and the corresponding recombinase polymerase amplification (RPA) primer sequence with the T7 promoter for the CRISPR assay. Twenty-five clinical isolates and 43 bronchoalveolar lavage fluid (BALF) remaining from routine examinations of patients with confirmed pulmonary aspergillosis were collected to further validate the CRISPR assay.
Results:
No amplification signal was observed when genomic DNA from closely clinically related Aspergillus species, such as Aspergillus flavus, Aspergillus niger, and Aspergillus terreus, as well as Monascus purpureus Went and Escherichia coli, was tested by this assay, and the detection limit for A. fumigatus was 3 copies in a single reaction system. Validation experiments using the 25 clinical isolates demonstrated 91.7% specificity for the A. fumigatus section, and the sensitivity was 100% when first-generation sequencing was used as the standard. There was no significant difference between the PCR and CRISPR methods (P = 1.0), and the diagnosis results of the two methods were consistent (Kappa = 0.459, P = 0.003).
Conclusion:
The study offers a new validated CRISPR/Cas13a technique for A. fumigatus detection, providing a simple, rapid and affordable test that is ready for application in the diagnosis of A. fumigatus infection.
Insights
A new CRISPR/Cas13a diagnostic method accurately detects Aspergillus fumigatus. This rapid, sensitive, and specific test offers a promising tool for diagnosing fungal infections.
Area of Science:
- Molecular Biology
- Diagnostic Microbiology
Background:
- Rapid and reliable diagnostics for Aspergillus fumigatus infection are crucial.
- CRISPR-Cas13a shows high sensitivity and specificity for viral detection but its use for fungal pathogens is unexplored.
Purpose of the Study:
- To develop and validate a sensitive and specific CRISPR/Cas13a-based method for detecting Aspergillus fumigatus.
Main Methods:
- Designed CRISPR-derived RNA (crRNA) and recombinase polymerase amplification (RPA) primers targeting the internal transcribed spacer (ITS) region of A. fumigatus.
- Validated the assay using 25 clinical isolates and 43 bronchoalveolar lavage fluid (BALF) samples from patients with pulmonary aspergillosis.
Main Results:
- The assay demonstrated high specificity, with no cross-reactivity to related Aspergillus species or other microbes.
- Achieved a detection limit of 3 copies for A. fumigatus, with 100% sensitivity and 91.7% specificity in clinical isolates.
- CRISPR/Cas13a results were consistent with PCR, showing no significant difference (P=1.0).
Conclusions:
- A validated CRISPR/Cas13a technique for A. fumigatus detection has been established.
- This method provides a simple, rapid, and affordable diagnostic test for A. fumigatus infections.
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