RPB0251

Pathogen Description

Target Pathogen Pathogen Name NCBI Taxonomy ID Order Family Genus Species Pathogen type
Klebsiella pneumoniae Klebsiella pneumoniae, Bacillus pneumoniae, Hyalococcus pneumoniae 573 Enterobacterales Enterobacteriaceae Klebsiella Klebsiella pneumoniae Bacterium

Primer Description

Primer Name Sequence(5'-3') Length(bp) Primer Final Concentration(μM） GC Content（%） Predicted Melting Temperature(℃） Molecular Weight(g/moles) Positions in GenBank accession number
Kpn-RPA-F CTGTACGTCGTGTTGCCCGGCGGTGGTGTTTC 32 10 μM 62.5 71.78 9861.39 \
Kpn-RPA-R GCGAAGTACCATGCCCGGCCATCCACATTTGC 32 10 μM 59.38 70.64 9730.36 \

Gene Description

Target Gene GenBank ID
rcsA gene \

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
the immediate, sensitive and efficient detection of K. pneumoniae. EXORCA (EXtraction-free One-pot RPA-CRISPR\Cas12a assay) \ 30min 39 °C CRISPR\Cas12a 10CFU/μL 100 % 100 %

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2025 An extraction-free one-pot assay for rapid detection of Klebsiella pneumoniae by combining RPA and CRISPR\Cas12a Jinyu Fu,Rurong Mo,Ziyao Li,Shijie Xu,Xiyu Cheng,Binghuai Lu,Shuobo Shi Biosensors and Bioelectronics 39244837 10.1016/j.bios.2024.116740

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

Author(s):

Jinyu Fu,Rurong Mo,Ziyao Li,Shijie Xu,Xiyu Cheng,Binghuai Lu,Shuobo Shi

Journal:

Biosensors and Bioelectronics

Year:

2025

Abstract:

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.