New Gene Test Identifies Bloodstream Pathogens Without Need for Amplification

New Delhi: A team of scientists has unveiled a gene-based diagnostic technology capable of detecting multi-drug-resistant bacteria and other pathogens in the bloodstream at extremely low concentrations—without the need for nucleic acid amplification. The innovative research was recently published in the journal Proceedings of the National Academy of Sciences of the United States of America (PNAS).

The team, led by Professor Rashid Bashir of the University of Illinois Grainger College of Engineering, focused on improving the sensitivity and speed of pathogen detection. Traditional CRISPR/Cas-based diagnostic tests rely on guide RNAs to identify pathogen DNA or RNA, which then activate Cas enzymes to cleave reporter nucleic acids. This cleavage produces a fluorescent signal, indicating the presence of a pathogen. However, these conventional single-unit CRISPR tests struggle to detect low levels of pathogens without incorporating a pre-amplification step, which adds complexity and time.

To overcome this limitation, Bashir’s team developed a novel approach called CRISPR-Cascade. This system integrates two CRISPR/Cas units working in tandem. The first unit uses a guide RNA tailored to a specific pathogen’s genetic material alongside a Cas protein. Once the Cas enzyme cleaves specially designed nucleic acids introduced into the system, fragments from these nucleic acids activate a second CRISPR/Cas unit. This activation sets off a positive feedback loop, significantly enhancing the signal-to-noise ratio and eliminating the need for amplification.

In laboratory tests, the CRISPR-Cascade system demonstrated remarkable sensitivity, detecting multi-drug-resistant Staphylococcus aureus DNA at concentrations much lower than what is achievable with standard single Cas-based tests. Additionally, the technology delivered clear "yes/no" results for the presence of any one pathogen in samples spiked with four common bloodstream pathogens.

The researchers highlight that this advancement could pave the way for the development of next-generation CRISPR-based diagnostic tests. These tests promise rapid, highly sensitive pathogen detection in clinical settings, providing results in just minutes without requiring complex amplification procedures.

This breakthrough holds significant potential for improving early diagnosis of bloodstream infections, including those caused by antibiotic-resistant bacteria, ultimately contributing to better patient outcomes and more efficient infection control.