RPB0239

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
NDM-RAA-F2 AATTCTAATACGACTCACTATAGGGTGRRCGCGCTGCATGCGGCGGGGATTGCGA 55 1 mM 54.55 75.63 17056.08 \
NDM-RAA-R2 CCATTGGCGGCGAAAGTCAGGCTGTGTTGC 30 1 mM 60 70 9279.05 \

Gene Description

Target Gene GenBank ID
blaNDM gene \

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
PCR\RAA-CRISPR assays for the detection of blaKPC and blaNDM carbapenemase genes, as well as KP, to facilitate the detection of CRKP. RAA-CRISPR\Cas13a lateral flow strip Primer 3 20 min 37°C fluorescence signal readout and the lateral flow strip readout 101copies/μL 100 % 100 %

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2024 CRISPR\Cas13-assisted carbapenem-resistant Klebsiella pneumoniae detection Yaling Cao,Yuan Tian,Jing Huang,Ling Xu,Zihao Fan,Zhenzhen Pan,Sisi Chen,Yao Gao,Linlin Wei,Sujun Zheng,Xiangying Zhang,Yanhua Yu,Feng Ren Journal of Microbiology,Immunology and Infection 37963801 10.1016/j.jmii.2023.10.010

CRISPR\Cas13-assisted carbapenem-resistant Klebsiella pneumoniae detection

Author(s):

Yaling Cao,Yuan Tian,Jing Huang,Ling Xu,Zihao Fan,Zhenzhen Pan,Sisi Chen,Yao Gao,Linlin Wei,Sujun Zheng,Xiangying Zhang,Yanhua Yu,Feng Ren

Journal:

Journal of Microbiology,Immunology and Infection

Year:

2024

Abstract:

Background/purpose: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is capable of causing serious community and hospital-acquired infections. However, currently, the identification of CRKP is complex and inefficient. Hence, this study aimed to develop methods for the early and effective identification of CRKP to allow reasonable antimicrobial therapy in a timely manner. Methods: K. pneumoniae (KP)-, K. pneumoniae carbapenemase (KPC)- and New Delhi metallo-β-lactamase (NDM)- specific CRISPR RNAs (crRNAs), polymerase chain reaction (PCR) primers and recombinase-aided amplification (RAA) primers were designed and screened in conserved sequence regions. We established fluorescence and lateral flow strip assays based on CRISPR/Cas13a combined with PCR and RAA, respectively, to assist in the detection of CRKP. Sixty-one clinical strains (including 51 CRKP strains and 10 carbapenem-sensitive strains) were collected for clinical validation. Results: Using the PCR-CRISPR assay, the limit of detection (LOD) for KP and the blaKPC and blaNDM genes reached 1 copy/μL with the fluorescence signal readout. Using the RAA-CRISPR assay, the LOD could reach 101 copies/μL with both the fluorescence signal readout and the lateral flow strip readout. Additionally, the positivity rates of CRKP-positive samples detected by the PCR/RAA-CRISPR fluorescence and RAA-CRISPR lateral flow strip methods was 92.16% (47/51). The sensitivity and specificity reached 100% for KP and blaKPC and blaNDM gene detection. For detection in a simulated environmental sample, 1 CFU/cm2 KP could be detected. Conclusion: We established PCR/RAA-CRISPR assays for the detection of blaKPC and blaNDM carbapenemase genes, as well as KP, to facilitate the detection of CRKP.