Dataset Information
Development and Evaluation of Rapid and Accurate CRISPR/Cas13-Based RNA Diagnostics for Pneumocystis jirovecii Pneumonia.
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ABSTRACT: Background
Pneumocystis jirovecii can result in a serious pulmonary infection, Pneumocystis jirovecii pneumonia, in immunocompetent hosts. The diagnosis of Pneumocystis jirovecii pneumonia has long been a major clinical concern, and there are limitations with the currently utilized immunostaining and polymerase chain reaction diagnosis/detection technologies (e.g., insufficient sensitivity and accuracy). Hence, we sought to establish a rapid and RNA-specific transcription mediated amplification and CRISPR/Cas13a-based diagnostics targeted P. jirovecii-mitochondrial large subunit ribosomal RNA.Methods
The procedure of the diagnostics included amplification of the extracted RNA samples by transcription mediated amplification, followed by CRISPR/Cas13 detection, and ultimately, the judgment of the results after 30 minutes of fluorescence signal. Later, the diagnostic performance of the CRISPR/Cas13-based diagnostics were tested on the 62 surplus clinical samples.Results
This CRISPR/Cas13-based diagnostics achieved limits of detection of approximately 2 copies/µL transcribed RNA templates, with no cross reaction to other respiratory pathogens, including bacteria and fungi. Similar to in-house quantitative real-time polymerase chain reaction, CRISPR/Cas13-based diagnostics was still positive in 243-fold diluted bronchial alveolar lavage fluid. A preliminary evaluation of 62 surplus bronchial alveolar lavage fluid samples from patients suspected of Pneumocystis jirovecii pneumonia showed that CRISPR/Cas13-based diagnostics achieved a 78.9% sensitivity and a 97.7% specificity in the diagnosis of Pneumocystis jirovecii pneumonia.Conclusion
Our study demonstrates that the CRISPR/Cas13-based diagnostics technique has good performance for the accurate and specific diagnosis of Pneumocystis jirovecii pneumonia.
SUBMITTER: Zhan Y
PROVIDER: S-EPMC9240425 | biostudies-literature | 2022
REPOSITORIES: biostudies-literature
Publications
Development and Evaluation of Rapid and Accurate CRISPR/Cas13-Based RNA Diagnostics for <i>Pneumocystis jirovecii</i> Pneumonia.
Frontiers in cellular and infection microbiology 20220615
<h4>Background</h4><i>Pneumocystis jirovecii</i> can result in a serious pulmonary infection, <i>Pneumocystis jirovecii</i> pneumonia, in immunocompetent hosts. The diagnosis of <i>Pneumocystis jirovecii</i> pneumonia has long been a major clinical concern, and there are limitations with the currently utilized immunostaining and polymerase chain reaction diagnosis/detection technologies (<i>e.g.</i>, insufficient sensitivity and accuracy). Hence, we sought to establish a rapid and RNA-specific t ...[more]