Bioengineering professor and The Grainger College of Engineering’s Dean, Rashid Bashir, led a team of researchers in a project that’s resulted in new technology that offers rapid, highly sensitive detection of multidrug-resistant bacteria and other pathogens at low concentrations.
This research was featured in an article in the Proceedings of the National Academy of Sciences of the United States of America (PNAS).
Researchers designed a CRISPR-based test that rapidly detects low levels of pathogen genetic material in blood. This is done without the need for nucleic acid amplification.
In CRISPR/Cas-based diagnostic tests, guide RNAs bind pathogen DNA or RNA, triggering Cas enzymes to become active and cleave the reporter nucleic acids that fluoresce when cleaved. However, the single CRISPR-based technique does not detect pathogens at low levels without a preamplification step.
Amplification-free CRISPR-Cascade diagnostics
Bashir’s team created a CRISPR-based diagnostic test that bypasses that amplification step by combining two CRISPR/Cas units in a complex called CRISPR-Cascade. One unit contains a guide RNA specific for a pathogen nucleic acid of choice and a Cas protein. When the Cas cleaves specially engineered nucleic acids that are added to the system, parts of the nucleic acids are free to bind and activate a second CRISPR/Cas, triggering a positive feedback loop that results in a high signal-to-noise ratio.
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The test demonstrated unprecedented sensitivity. It also detected multidrug-resistant Staphylococcus aureus DNA without prior amplification at concentrations that were orders of magnitude lower than the limit of a test using a single Cas. The test provided a simple “yes/no” result for the presence of any one pathogen in samples spiked with four common bloodstream pathogens.
The researchers said these results could be used toward developing highly sensitive CRISPR-based diagnostic tests that can detect pathogens in minutes without nucleic acid amplification.
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