Breakthrough Tech Identifies Pathogens in 3 Hours

Abstract

Fast and accurate identification of pathogenic microbes in patient samples is crucial for the timely treatment of acute infectious diseases such as sepsis. The fluorescence in situ hybridization (FISH) technique allows the rapid detection and identification of microbes based on their variation in genomic sequence without time-consuming culturing or sequencing. However, the recent explosion of microbial genomic data has made it challenging to design an appropriate set of probes for microbial mixtures. We developed a novel set of peptide nucleic acid (PNA)-based FISH probes with optimal target specificity by analyzing the variations in 16S ribosomal RNA sequence across all bacterial species. Owing to their superior penetration into bacteria and higher mismatch sensitivity, the PNA probes distinguished seven bacterial species commonly observed in bacteremia with 96-99.9% accuracy using our optimized FISH procedure. Detection based on Förster resonance energy transfer (FRET) between pairs of adjacent binding PNA probes eliminated crosstalk between species. Rapid sequential species identification was implemented, using chemically cleavable fluorophores, without compromising detection accuracy. Owing to their outstanding accuracy and enhanced speed, this set of techniques shows great potential for clinical use.

A research team, affiliated with UNIST has developed a diagnostic technology capable of identifying infectious pathogens with nearly 100% accuracy in under three hours. This method is significantly faster and more accurate than traditional bacterial culture and polymerase chain reaction (PCR) analysis, and it holds promise for reducing mortality rates in critical conditions such as sepsis, where timely administration of antibiotics is crucial.

The joint team of professors-Hajun Kim, Taejun Kwon, and Joohun Kang-from the Department of Biomedical Engineering at UNIST has unveiled a novel diagnostic technique that utilizes artificially designed polymers known as peptide nucleic acid (PNA) as probes. The reported fluorescence in situ hybridization (FISH) technique works by detecting fluorescent signals generated when probe molecules bind to specific genetic sequences in bacteria.

This innovative FISH method employs two PNA molecules simultaneously. By analyzing the genomic sequences of 20,000 bacterial species, the research team designed PNA sequences that specifically target the ribosomal RNA of particular species. PNA exhibits a higher sensitivity to sequence mismatches compared to conventional DNA-based probes and demonstrates superior penetration through bacterial cell walls. Furthermore, the requirement for both PNA molecules to bind to their target site before generating a signal significantly reduces the likelihood of crosstalk, thereby enhancing accuracy in situations involving multiple overlapping bacterial strains.

In tests, the technology successfully detected seven bacterial species-including E. coli, Pseudomonas aeruginosa, and Staphylococcus aureus-showing over 99% accuracy for all species except Staphylococcus aureus, which was detected with an accuracy of 96.3%. The method's effectiveness was further validated in mixed bacterial samples. Both Enterococcus and E. coli were detected with over 99% accuracy when tested together.

The approach utilizing two PNA molecules is based on Förster Resonance Energy Transfer (FRET). This principle involves energy transfer from one PNA molecule to another when they are in close proximity, allowing for the measurement of the fluorescence emitted by the recipient molecule.

Professor Kim noted, "This method will aid in the diagnosis of infections requiring immediate antibiotic treatment, such as sepsis, urinary tract infections, and pneumonia, while also helping to reduce unnecessary antibiotic usage." The research team plans to conduct further experiments using blood samples taken from actual patients to explore clinical applications.

The research findings were published in the journal, Biosensors and Bioelectronics on March 1, 2025. The study features contributions from Dr. Sungho Kim and Dr. Hwi Hyun from the Department of Biomedical Engineering at UNIST as first authors, and it was supported by the National Research Foundation of Korea (NRF), the Institute for Basic Science (IBS), the Korea National Institute of Health (NIH), and UNIST.

Journal Reference

Sungho Kim, Hwi Hyun, Jae-Kyeong Im, et al., "Fast and accurate multi-bacterial identification using cleavable and FRET-based peptide nucleic acid probes," Biosens. Bioelectron., (2025).