Rapid 65-min SYBR-Green PCR Assay for Carbapenem Resistant Klebsiella and Acinetobacter Detection

BUKAVAZ Ş., Gungor K., KUNDURACILAR H., YULUĞKURAL Z.

Microorganisms, cilt.13, sa.11, 2025 (SCI-Expanded, Scopus) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 13 Sayı: 11
  • Basım Tarihi: 2025
  • Doi Numarası: 10.3390/microorganisms13112590
  • Dergi Adı: Microorganisms
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, BIOSIS, Directory of Open Access Journals
  • Anahtar Kelimeler: Acinetobacter baumannii, Klebsiella pneumoniae, SYBR-Green PCR
  • Trakya Üniversitesi Adresli: Evet

Özet

This study developed a rapid and reliable SYBR-Green semiplex PCR assay for simulta-neous detection of major carbapenem resistance genes in Klebsiella pneumoniae and Acinetobacter baumannii. Two primer sets were used: one to detect blaKPC, blaNDM-1, and blaOXA-48 genes in K. pneumoniae and blaOXA-23 in A. baumannii, and another to amplify conserved 16S rRNA gene regions as internal controls. The intra- and inter-assay coeffi-cient of variation ranged from 0.03% to 3.8%. Standard curves exhibited excellent linearity across six logarithmic scales (101–106 DNA copies/µL), with detection limits of 10–102 DNA copies/mL. Melting temperatures (Tm) were: 88.85 °C (KPIC), 90.65 °C (NDM-1), 89.45 °C (KPC), 84.23 °C (OXA-48), 87.81 °C (OXA-23), and 80.67 °C (ABIC). The SYBR-Green Semiplex PCR assay offers a rapid (65 min turnaround), cost-effective, and sensitive method for early detection of carbapenem-resistant pathogens, enabling timely targeted therapy and improved infection control by potentially reducing empirical antibiotic use before culture confirmation.