Development and evaluation of a duplex real-time multienzyme isothermal rapid amplification assay for the detection of hypervirulent Klebsiella pneumoniae in clinical spiked blood specimens
Year: 2024
Type: article
Abstract: Objectives Our objective was to establish a rapid and precise method for detecting hypervirulent Klebsiella pneumoniae (hvKP) by utilizing a duplex real-time multienzyme isothermal rapid amplification (real-time MIRA) and to evaluate its performance in clinical spiked blood specimens. Methods The research comprised two phases: an initial pilot study to establish the methodology and a clinical validation study to assess its effectiveness. In the pilot phase, we designed specific primers and probes targeting the hvKP pg344 and incA genes and subsequently developed a duplex real-time MIRA assay to evaluate its detection limits, specificity, and efficiency. In the clinical validation phase, we analyzed thirty-three spiked blood specimens using the duplex real-time MIRA assay. Results The duplex real-time MIRA assay demonstrated no cross-reactivity with other strains. Sensitivity experiments confirmed that the assay had a detection limit as low as 8 × 10 2 CFU per reaction for hvKP. The analysis of clinical spiked blood specimens indicated that the sensitivity and specificity of the duplex real-time MIRA assay were on par with those of duplex real-time PCR. Conclusions These findings confirm that the duplex real-time MIRA assay is a fast, straightforward, and dependable method for detecting hvKP.
Source: Heliyon
Institution: Chongqing Medical University
Language: English
Subfield: Biomedical Engineering
Field: Engineering
Domain: Physical Sciences
Open Access status: gold
Estimated APC paid: $2,100
OpenAlex ID: W4402031880