Development and application of an integrated full-sequence gene fluorescence quantitative PCR system for...

The spread of carbapenemase-producing Enterobacteriaceae (CPE) has become a global concern. Therefore, reliable methods for rapid carbapenemase detection are needed. Molecular methods are faster and offer higher sensitivity and specificity than phenotypic methods. However, conventional PCR assays often target partial sequences, which may miss variants or subtypes. This study aimed to develop a new full-sequence gene fluorescent quantitative PCR system for the detection of bla_KPC and bla_NDM genes in CPE.

We first designed full-sequence amplification primers targeting the bla_KPC and bla_NDM genes and established a full-sequence fluorescence quantitative PCR system. The specificity, sensitivity, and limit of detection of the system were subsequently evaluated. Finally, the performance of the system was assessed using 94 clinical carbapenem-resistant Enterobacterales (CRE) isolates (including 69 Klebsiella pneumoniae and 17 Escherichia coli).

The full-sequence fluorescence quantitative PCR system specifically amplified the bla_KPC and bla_NDM genes, with a limit of detection of 10²CFU/mL for both genes. The system exhibited strong robustness against interference. The sensitivity was 100.00% for both genes; the specificity was 91.30% for bla_KPC and 98.41% for bla_NDM. The Kappa coefficients for bla_KPC and bla_NDM were 0.9147 and 0.9761, respectively. The system was also capable of detecting strains harboring the bla_KPC-33 and bla_KPC-71 variants.

We developed a novel full-sequence fluorescence quantitative PCR detection system for the bla_KPC and bla_NDM genes, which are the carbapenemase genes with the highest detection rates in clinical settings. This detection method demonstrates high sensitivity, high specificity, and the ability to detect mutant genes.