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Simultaneous and Visual Detection of KPC and NDM Carbapenemase-Encoding Genes Using Asymmetric PCR and Multiplex
Wei Lai1, Yongjie Xu2, Lin Liu2
1School of Medical Laboratory, Guizhou Medical University, Guiyang 550004, Guizhou, China.
Abstract:
Carbapenem-resistant Enterobacteriaceae (CRE) infections constitute a threat to public health, and KPC and NDM are the major carbapenemases of concern. Rapid diagnostic tests are highly desirable in point-of-care (POC) and emergency laboratories with limited resources. Here, we developed a multiplex lateral flow assay based on asymmetric PCR and barcode capture probes for the simultaneous detection of KPC-2 and NDM-1. Biotinylated barcode capture probes corresponding to the KPC-2 and NDM-1 genes were designed and cast onto two different sensing zones of a nitrocellulose membrane after reacting with streptavidin to prepare a multiplex lateral flow strip. Streptavidin-coated gold nanoparticles (SA-AuNPs) were used as signal reporters. In response to the target carbapenemase genes, biotin-labelled ssDNA libraries were produced by asymmetric PCR, which bond to SA-AuNPs via biotin and hybridise with the barcode capture probe via a complementary sequence, thereby bridging SA-AuNPs and the barcode capture probe to form visible red lines on the detection zones. The signal intensities were proportional to the number of resistance genes tested. The strip sensor showed detection limits of 0.03 pM for the KPC-2 and 0.07 pM for NDM-1 genes, respectively, and could accurately distinguish between KPC-2 and NDM-1 genes in CRE strains. For the genotyping of clinical isolates, our strip exhibited excellent consistency with real-time fluorescent quantitative PCR and gene sequencing. Given its simplicity, cost-effectiveness, and rapid analysis accomplished by the naked eye, the multiplex strip is promising auxiliary diagnostic tool for KPC-2 and NDM-1 producers in routine clinical laboratories.
Insights
A new multiplex lateral flow assay can rapidly detect carbapenemase-resistant Enterobacteriaceae (CRE) infections, specifically KPC-2 and NDM-1 genes. This cost-effective test aids in quick diagnosis at the point-of-care.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Biotechnology
Background:
- Carbapenem-resistant Enterobacteriaceae (CRE) pose a significant public health threat.
- KPC and NDM are the most concerning carbapenemase enzymes.
- Rapid point-of-care diagnostics are crucial for resource-limited settings.
Purpose of the Study:
- To develop a multiplex lateral flow assay for simultaneous detection of KPC-2 and NDM-1 genes.
- To provide a rapid, cost-effective diagnostic tool for KPC and NDM carbapenemases.
Main Methods:
- Developed a multiplex lateral flow strip using asymmetric PCR and barcode capture probes.
- Utilized streptavidin-coated gold nanoparticles (SA-AuNPs) as signal reporters.
- Prepared biotinylated ssDNA libraries that hybridize to capture probes on the strip.
Main Results:
- The assay achieved detection limits of 0.03 pM for KPC-2 and 0.07 pM for NDM-1.
- The strip accurately distinguished between KPC-2 and NDM-1 genes in CRE strains.
- Demonstrated excellent consistency with real-time PCR and gene sequencing for clinical isolates.
Conclusions:
- The developed multiplex strip is a promising auxiliary diagnostic tool for KPC-2 and NDM-1 producers.
- Its simplicity, cost-effectiveness, and rapid visual detection are suitable for routine clinical laboratories.
- Facilitates faster identification of carbapenemase-producing Enterobacteriaceae.
