CRISPR-Based Diagnostic Test Detects Pathogens in Blood Without Amplification

Traditional molecular diagnostics often require nucleic acid preamplification, which increases both time and cost. Now, a new technology enables rapid and highly sensitive detection of multi-drug-resistant bacteria and other pathogens, even at low concentrations.

Researchers from the University of Illinois Grainger College of Engineering (Urbana, IL, USA) have developed a CRISPR-based diagnostic test capable of rapidly detecting low levels of pathogen genetic material in blood samples, without the need for nucleic acid amplification. In CRISPR/Cas diagnostic tests, guide RNAs bind to pathogen DNA or RNA, triggering the activation of Cas enzymes that cleave reporter nucleic acids, which fluoresce when cleaved. However, traditional CRISPR-based techniques struggle to detect pathogens at low concentrations without a preamplification step. To address this, the team developed a CRISPR-based test that eliminates the need for amplification by combining two CRISPR/Cas systems into a complex known as CRISPR-Cascade. One part of the system contains a guide RNA specific to the pathogen's nucleic acid, along with a Cas protein. When the Cas protein cleaves specially engineered nucleic acids added to the system, portions of these nucleic acids can bind to and activate a second CRISPR/Cas unit, triggering a positive feedback loop that amplifies the signal, resulting in a high signal-to-noise ratio.

The research, published in the Proceedings of the National Academy of Sciences of the United States of America (PNAS), demonstrated unprecedented sensitivity for pathogen detection. It successfully detected multi-drug-resistant Staphylococcus aureus DNA without prior amplification at concentrations significantly lower than the detection limits of tests using a single Cas enzyme. The test provided a straightforward "yes/no" result for the presence of any one of four common bloodstream pathogens in spiked samples. The findings suggest that this approach could lead to the development of highly sensitive, rapid CRISPR-based diagnostic tests capable of detecting pathogens in minutes, without the need for nucleic acid amplification.