Image created by Dr. Michael J. Miller
Researchers at Ulsan National Institute of Science and Technology (UNIST) have developed an innovative molecular diagnostic technology capable of rapidly and accurately identifying the pathogens responsible for infections. This new method, which is faster and more cost-effective than conventional approaches, is expected to contribute to the early diagnosis of infectious diseases and help mitigate antibiotic misuse through clinical applications.
Fluorescence In Situ Hybridization (FISH) Enables Direct Pathogen Detection Without Culturing
A research team led by Professors Hajin Kim, Taejoon Kwon, and Jooheon Kang from UNIST’s Department of Biomedical Engineering announced the development of a Peptide Nucleic Acid (PNA)-based Fluorescence In Situ Hybridization (FISH) diagnostic technique.
FISH technology utilizes fluorescent probes that bind to specific gene sequences, allowing for the direct detection of bacterial genetic material. Unlike traditional Polymerase Chain Reaction (PCR) methods, FISH offers a faster and more cost-effective solution, capable of detecting infectious bacteria within just 12 hours, without the need for time-consuming culturing processes.
The newly developed method employs dual PNA molecules, which exhibit superior sensitivity compared to conventional DNA-based probes. Additionally, PNA’s ability to easily penetrate bacterial cell walls enhances its detection accuracy.
99% Detection Accuracy in Seven Bacterial Species
To ensure high specificity, the research team analyzed over 20,000 bacterial genome sequences and designed custom PNA sequences that selectively bind to ribosomal RNA of specific bacteria. Experimental validation demonstrated that the technology achieved over 99% detection accuracy for six bacterial species, including Escherichia coli and Pseudomonas aeruginosa, while detecting Staphylococcus aureus with 96.3% accuracy.
Furthermore, the method maintained high accuracy even in mixed bacterial environments. In samples containing both Enterococcus and E. coli, the detection accuracy remained above 99% for each bacterium, underscoring the reliability of this technology.
PNA-Based Förster Resonance Energy Transfer (FRET) Mechanism
The newly developed PNA-FISH diagnostic method is based on the Förster Resonance Energy Transfer (FRET) phenomenon. When two PNA molecules bind to the target genetic sequence, energy is transferred between them, generating a measurable fluorescent signal that enables precise pathogen identification.
Professor Hajin Kim emphasized the potential impact of this technology, stating, "This diagnostic method will significantly enhance the rapid identification of infectious diseases such as sepsis, urinary tract infections, and pneumonia, allowing for timely antibiotic treatment while reducing unnecessary antibiotic use."
Moving forward, the research team plans to conduct further clinical studies using blood samples from actual patients to assess the feasibility of real-world medical applications.
This research was published in the international journal Biosensors and Bioelectronics on March 1 and was supported by the National Research Foundation of Korea (NRF), Institute for Basic Science (IBS), Korea National Institute of Health (NIH), and UNIST.
Reference
Sungho Kim, Hwi Hyun, Jae-Kyeong Im, Min Seok Lee, Hwasoo Koh, Donghoon Kang, Si-Hyeong Nho, Joo H. Kang, Taejoon Kwon, Hajin Kim, Fast and accurate multi-bacterial identification using cleavable and FRET-based peptide nucleic acid probes, Biosensors and Bioelectronics, Volume 271, 2025, https://doi.org/10.1016/j.bios.2024.116950.
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.