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An extraction-free one-pot assay for rapid detection of Klebsiella pneumoniae by combining RPA and CRISPR/Cas12a.

Jinyu FuRurong MoZiyao LiShijie XuXiyu ChengBinghuai LuShuobo Shi
Published in: Biosensors & bioelectronics (2024)
Klebsiella pneumoniae poses a significant threat to global public health. Traditional clinical diagnostic methods, such as bacterial culture and microscopic identification, are not suitable for point-of-care testing. In response, based on the suboptimal protospacer adjacent motifs, this study develops an extraction-free one-pot assay, named EXORCA (EXtraction-free One-pot RPA-CRISPR/Cas12a assay), designed for the immediate, sensitive and efficient detection of K. pneumoniae. The EXORCA assay can be completed within approximately 30 min at a constant temperature and allows for the visualization of results either through a fluorescence reader or directly by the naked eye under blue light. The feasibility of the assay was evaluated using twenty unextracted clinical samples, achieving a 100% (5/5) positive predictive value and a 100% (15/15) negative predictive value in comparison to qPCR. These results suggest that the EXORCA assay holds significant potential as a point-of-care testing tool for the rapid identification of pathogens, such as K. pneumonia.
Keyphrases
  • klebsiella pneumoniae
  • crispr cas
  • high throughput
  • public health
  • multidrug resistant
  • escherichia coli
  • genome editing
  • intensive care unit
  • antimicrobial resistance
  • climate change
  • real time pcr
  • respiratory tract