Development and evaluation of a duplex real-time multienzyme isothermal rapid amplification assay for simultaneous detection of hypervirulent Klebsiella pneumoniae K1 and K2 serotypes in spiked blood samples

The emergence of hypervirulent Klebsiella pneumoniae (hvKP), particularly the K1 and K2 serotypes, presents a significant public health threat due to their increased virulence and resistance. Rapid and accurate detection of hvKP is crucial for effective clinical management. This study developed and evaluated a duplex real-time multienzyme isothermal rapid amplification (MIRA) assay for the simultaneous detection of hvKP K1 and K2 serotypes in spiked blood samples. The assay employed optimized primers and probes targeting specific capsular genes. Sensitivity and specificity were assessed using spiked blood specimens and compared to real-time PCR. The detection limit was 1 × 10³ CFU per reaction for both serotypes, with no cross-reactivity with non-hvKP K1/K2 strains. The assay demonstrated superior reproducibility and stability, offering faster detection and simplified infrastructure requirements compared to real-time PCR. These features make the duplex real-time MIRA assay a promising tool for clinical diagnostics and outbreak surveillance.