Development of RPA-Cas12a assay for rapid and sensitive detection of Pneumocystis jirovecii.
Pneumocystis carinii
/ genetics
Nucleic Acid Amplification Techniques
/ methods
Humans
Sensitivity and Specificity
Pneumonia, Pneumocystis
/ diagnosis
Molecular Diagnostic Techniques
/ methods
Endodeoxyribonucleases
/ genetics
CRISPR-Associated Proteins
/ genetics
DNA, Fungal
/ genetics
Recombinases
/ metabolism
Bacterial Proteins
Pneumocystis jirovecii
CRISPR-Cas12a
Detection
Recombinase polymerase amplification
Resource-limited settings
Journal
BMC microbiology
ISSN: 1471-2180
Titre abrégé: BMC Microbiol
Pays: England
ID NLM: 100966981
Informations de publication
Date de publication:
26 Aug 2024
26 Aug 2024
Historique:
received:
08
04
2024
accepted:
24
07
2024
medline:
27
8
2024
pubmed:
27
8
2024
entrez:
26
8
2024
Statut:
epublish
Résumé
Pneumocystis jirovecii is a prevalent opportunistic fungal pathogen that can lead to life-threatening Pneumocystis pneumonia in immunocompromised individuals. Given that timely and accurate diagnosis is essential for initiating prompt treatment and enhancing patient outcomes, it is vital to develop a rapid, simple, and sensitive method for P. jirovecii detection. Herein, we exploited a novel detection method for P. jirovecii by combining recombinase polymerase amplification (RPA) of nucleic acids isothermal amplification and the trans cleavage activity of Cas12a. The factors influencing the efficiency of RPA and Cas12a-mediated trans cleavage reaction, such as RPA primer, crRNA, the ratio of crRNA to Cas12a and ssDNA reporter concentration, were optimized. Our RPA-Cas12a-based fluorescent assay can be completed within 30-40 min, comprising a 25-30 min RPA reaction and a 5-10 min trans cleavage reaction. It can achieve a lower detection threshold of 0.5 copies/µL of target DNA with high specificity. Moreover, our RPA-Cas12a-based fluorescent method was examined using 30 artificial samples and demonstrated high accuracy with a diagnostic accuracy of 93.33%. In conclusion, a novel, rapid, sensitive, and cost-effective RPA-Cas12a-based detection method was developed and demonstrates significant potential for on-site detection of P. jirovecii in resource-limited settings.
Identifiants
pubmed: 39187803
doi: 10.1186/s12866-024-03440-z
pii: 10.1186/s12866-024-03440-z
doi:
Substances chimiques
Endodeoxyribonucleases
EC 3.1.-
Cas12a protein
EC 3.1.-
CRISPR-Associated Proteins
0
DNA, Fungal
0
Recombinases
0
Bacterial Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
314Informations de copyright
© 2024. The Author(s).
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