Breakthrough Diagnostic Technology Identifies Bacterial Infections with Almost 100% Accuracy within Three Hours

The fluorescence in situ hybridization (FISH) technique facilitates the swift detection and identification of microbes by leveraging differences in their genomic sequences, without the need for time-consuming culturing or sequencing. However, the growing volume of microbial genomic data has made it increasingly difficult to design appropriate probes for microbial mixtures. Now, a new set of peptide nucleic acid (PNA)-based FISH probes has been developed, offering optimal target specificity by analyzing variations in the 16S ribosomal RNA sequence across bacterial species. Due to their superior ability to penetrate bacteria and higher sensitivity to mismatches, these PNA probes successfully identified seven bacterial species commonly associated with bacteremia with an accuracy ranging from 96% to 99.9% using the optimized FISH technique. Detection is enhanced by Förster resonance energy transfer (FRET) between adjacent binding PNA probes, which prevents cross-reactivity between species. This approach allows for rapid, sequential identification of bacterial species, utilizing chemically cleavable fluorophores, without sacrificing accuracy. Thanks to their exceptional accuracy and speed, these techniques hold significant promise for clinical applications.

A team of researchers from UNIST (Ulsan, Republic of Korea) has developed a diagnostic method capable of identifying infectious pathogens with nearly 100% accuracy in under three hours. This method is far faster and more accurate than traditional bacterial culture and polymerase chain reaction (PCR) analysis, offering potential to reduce mortality rates in critical conditions such as sepsis, where the prompt administration of antibiotics is essential. In their study, published in Biosensors and Bioelectronics, the researchers introduced a new diagnostic approach that uses PNA-based probes to detect pathogens. The FISH technique works by detecting fluorescent signals generated when the probe molecules bind to specific bacterial genetic sequences. This innovative method utilizes two PNA molecules at once, with the researchers designing PNA sequences that specifically target the ribosomal RNA of particular bacterial species by analyzing the genomic sequences of 20,000 species.

PNA exhibits greater sensitivity to sequence mismatches compared to conventional DNA-based probes, and it has 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 risk of crosstalk, thereby enhancing accuracy when multiple bacterial strains overlap. In tests, the technology successfully detected seven bacterial species—including E. coli, Pseudomonas aeruginosa, and Staphylococcus aureus—with over 99% accuracy for all species except Staphylococcus aureus, which was detected with an accuracy of 96.3%. The method’s effectiveness was also validated in mixed bacterial samples, where Enterococcus and E. coli were identified with over 99% accuracy when tested together. The research team plans further experiments using blood samples from actual patients to explore the clinical applications of this method.

“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,” said Professor Hajun Kim from the Department of Biomedical Engineering at UNIST.

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