Singleplex real-time PCR and duplex PCR platform for the rapid detection of hypervirulent Klebsiella pneumoniae

Area of Science:

• Microbiology and Infectious Diseases
• Molecular Diagnostics
• Public Health Microbiology

Background:

• Hypervirulent Klebsiella pneumoniae (HvKP) is a significant zoonotic pathogen causing severe infections with high mortality.
• Current identification methods, like the string test, are laborious and time-consuming, hindering rapid public health response.
• A need exists for swift, accurate diagnostic tools to track HvKP spread and implement timely control measures.

Purpose of the Study:

• To develop rapid, sensitive, and specific diagnostic assays for identifying HvKP strains.
• To utilize real-time PCR with SYBR Green I for efficient detection based on specific HvKP biomarkers.

Main Methods:

• Development of SYBR Green I-based singleplex real-time PCR assays targeting HvKP biomarkers: iroB, iucA, peg-344, and rmpA.
• Optimization of duplex PCR melting curve assays to enhance diagnostic efficiency and reduce costs.
• Evaluation of assay linearity, limit of detection (LOD), and specificity against other bacterial pathogens.

Main Results:

• Singleplex PCR assays demonstrated high linearity (R² > 0.99) and an LOD of 10³ CFU/mL (10 CFU/reaction).
• Duplex PCR assays achieved LODs ranging from 10⁴ to 10⁵ CFU/mL, offering improved efficiency.
• All developed assays exhibited high specificity, accurately distinguishing HvKP from other bacterial species.

Conclusions:

• The developed SYBR Green I-based real-time PCR assays provide rapid, sensitive, and specific identification of HvKP.
• These assays serve as valuable diagnostic tools for public health surveillance and control of HvKP outbreaks.
• The duplex PCR format offers a cost-effective and efficient approach for routine diagnostic testing.