RPB0402

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
KPC-F CTTCAG CAACAA ATTGGC GGCGG CGT TATC 30 \ 53.33 66.3 9207.03 \
KPC-R CCACTGTGCAGC TCATTCAAG GGC TTTCTT 30 \ 50 64.81 9123.97 \

Gene Description

Target Gene GenBank ID
bla KPC AY034847.1

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
the RPA-Cas13a system is a simple and one-hour efficient technology for the detection of a potentially fatal antibiotic resistance gene. RPA-Cas13a Primer Premier 5 30 min 39°C Cas13a 2.5 copies/μL 0.965 1

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2023 Rapid Detection of blaKPC in Carbapenem-Resistant Enterobacterales Based on CRISPR\Cas13a Mingjun Liang,Bin Xiao,Lidan Chen,Xiaoyan Huang,Jinchao Li,Zhenzhan Kuang,Xinping Chen,Xiuna Huang,Zhaohui Sun,Linhai Li Current Microbiology 37737960 10.1007/s00284-023-03457-z

Rapid Detection of blaKPC in Carbapenem-Resistant Enterobacterales Based on CRISPR\Cas13a

Author(s):

Mingjun Liang,Bin Xiao,Lidan Chen,Xiaoyan Huang,Jinchao Li,Zhenzhan Kuang,Xinping Chen,Xiuna Huang,Zhaohui Sun,Linhai Li

Journal:

Current Microbiology

Year:

2023

Abstract:

Klebsiella pneumoniae carbapenemase (KPC) is a crucial enzyme that causes carbapenem resistance in Enterobacterales, and infections by these "superbugs" are extremely challenging to treat. Therefore, there is a pressing need for a rapid and accurate KPC detection test to control the prevalence of carbapenem-resistant Enterobacterales (CREs). In this study, we established a novel method for detection of blaKPC, the gene responsible for encoding KPC, based on a recombinase polymerase amplification (RPA) and a CRISPR/Cas13a reaction coupled to fluorophore activation (termed RPA-Cas13a assay). We carefully selected a pair of optimal amplification primers for blaKPC and achieved a lower limit of detection of approximately 2.5 copies/μL by repeatedly amplifying a recombinant plasmid containing blaKPC. The RPA-Cas13a assay demonstrated a sensitivity of 96.5% and specificity of 100% when tested on 57 blaKPC-positive CRE strains, which were confirmed by DNA sequencing. Moreover, in 311 sputum samples, the theoretical antibiotic resistance characteristics of blaKPC-positive strains obtained by the RPA-Cas13a assay were highly consistent with the results of antibiotic susceptibility test (Kappa = 0.978 > 0.81, P < 0.01). In conclusion, the RPA-Cas13a system is a simple and one-hour efficient technology for the detection of a potentially fatal antibiotic resistance gene.