Ultrasensitive Pathogen Detection Without Nucleic Acid Amplification

Rashid Bashir, a Bioengineering Professor and Dean of the Grainger College of Engineering oversaw a group of researchers in a project that produced new technology that provides quick, extremely sensitive detection of bacteria and other pathogens at low concentrations that are resistant to multiple drugs.

The journal Proceedings of the National Academy of Sciences of the United States of America Published this study.

Researchers created a CRISPR-based test to quickly identify trace amounts of pathogen genetic material in blood. Nucleic acid amplification is not required for this.

When guide RNAs attach to pathogen DNA or RNA, Cas enzymes become active and cleave the reporter nucleic acids, which fluoresce when cleaved, in CRISPR/Cas-based diagnostic tests. However, without a preamplification step, the CRISPR-based method alone is unable to identify pathogens at low concentrations.

Bashir's group combined two CRISPR/Cas units into a complex known as CRISPR-Cascade to develop a CRISPR-based diagnostic test that avoids that amplification step. One unit includes a Cas protein and a guide RNA tailored to a particular pathogen nucleic acid.

A positive feedback loop that produces a high signal-to-noise ratio is triggered when the Cas cleaves specially engineered nucleic acids that are added to the system. Parts of the nucleic acids are then free to bind and activate a second CRISPR/Cas.

The test's sensitivity was unprecedented. Additionally, it found multi-drug-resistant Staphylococcus aureus DNA at concentrations orders of magnitude lower than the limit of a test employing a single Cas without the need for prior amplification.

When four common bloodstream pathogens were added to samples, the test yielded a straightforward “yes/no” response for the presence of any one pathogen.

According to the researchers, these findings could be applied to the creation of extremely sensitive CRISPR-based diagnostic tests that do not require nucleic acid amplification and can identify infections in a matter of minutes.