A Multiplex Recombinase‐Aided qPCR Assay for Highly Sensitive and Rapid Detection of khe, blaKPC ‐2, and blaNDM ‐1 Genes in Klebsiella pneumoniae
Shao‐wei Hua, Jie Wang, Zi‐jin Zhao, Feng‐yu Tian, Meng Zhao, Yu‐xin Wang, Rui‐qing Zhang, Zhi‐qiang Han, Shi‐jue Gao, Xiao‐na Lv, Hong‐yi Li, Xin‐xin Shen, Xue‐jun Ma, Zhi‐shan Feng - Microbiology (medical)
- Biochemistry (medical)
- Medical Laboratory Technology
- Clinical Biochemistry
- Public Health, Environmental and Occupational Health
- Hematology
- Immunology and Allergy
ABSTRACT
Objective
This study aimed to establish a highly sensitive and rapid single‐tube, two‐stage, multiplex recombinase‐aided qPCR (mRAP) assay to specifically detect the khe, blaKPC‐2, and blaNDM‐1 genes in Klebsiella pneumoniae.
Methods
mRAP was carried out in a qPCR instrument within 1 h. The analytical sensitivities of mRAP for khe, blaKPC‐2, and blaNDM‐1 genes were tested using recombinant plasmids and dilutions of reference strains. A total of 137 clinical isolates and 86 sputum samples were used to validate the clinical performance of mRAP.
Results
mRAP achieved the sensitivities of 10, 8, and 14 copies/reaction for khe, blaKPC‐2, and blaNDM‐1 genes, respectively, superior to qPCR. The Kappa value of qPCR and mRAP for detecting khe, blaKPC‐2, and blaNDM‐1 genes was 1, 0.855, and 1, respectively (p < 0.05).
Conclusion
mRAP is a rapid and highly sensitive assay for potential clinical identification of khe, blaKPC‐2, and blaNDM‐1 genes in K. pneumoniae.