Rapid Micro Methods Blog CRISPR-Cascade Test Detects Bloodstream Infections in Minutes Without Amplification

Image created by Dr. Michael J. MillerUniversity of Illinois at Urbana-Champaign researchers have developed a CRISPR-based diagnostic tool capable of detecting bloodstream infections in minutes without the need for nucleic acid amplification. The CRISPR-Cascade assay achieves attomolar sensitivity and incorporates an OR-gated logic function to identify multiple pathogens simultaneously through DNA from pathogens associated with bloodstream infections.Bloodstream infections require rapid identification to prevent complications, yet standard diagnostic methods rely on polymerase chain reaction (PCR) and isothermal amplification techniques that have built-in processing times. CRISPR-based detection tools such as SHERLOCK and DETECTR have improved specificity but continue to depend on amplification, limiting their turnaround time and practicality in clinical settings.In the study, "Amplification-free, OR-gated CRISPR-Cascade reaction for pathogen detection in blood samples," published in the Proceedings of the National Academy of Sciences, researchers conducted a laboratory-based investigation to determine whether a CRISPR-driven feedback loop could detect pathogenic DNA at ultra-low concentrations without amplification.Their CRISPR-Cascade reaction utilizes two ribonucleoprotein complexes, T1 and T2, to achieve amplification-free detection of pathogenic DNA. Upon target recognition, Cas12a in the T1 complex cleaves blocked nucleic acids, triggering the release of switchable caged RNA that activates more Cas12a enzymes in the T2 complex. This self-sustaining feedback loop enables rapid fluorescence-based detection at attomolar sensitivity within 10 minutes.Optimizations in reaction temperature (33°C), steric hindrance strategies, and enzyme kinetics improved specificity and reduced nonspecific signal interference.In testing, the assay successfully identified Methicillin-Sensitive Staphylococcus aureus (MSSA), Methicillin-Resistant Staphylococcus aureus (MRSA), Escherichia coli (E. coli), and Hepatitis B Virus in pathogen DNA-spiked blood samples within ten minutes. An OR-gated logic function enabled simultaneous detection of multiple pathogens in a single reaction, providing a rapid and scalable approach for clinical applications.Comparisons with existing methods highlight the accelerated timeline. Traditional culturing with PCR can take 24-72 hours. PCR methods without culturing run for 3-6 hours. Rapid PCR-based methods exist that take around 1 hour.CRISPR-Cascade, including sample preparation, requires about half an hour, making it the fastest available bloodstream infection diagnostic. There is also the potential to improve sample prep further and approach an under-15-minute mark. Potential applications include infectious disease diagnostics, point-of-care testing, and personalized medicine.ReferenceJongwon Lim et al, Amplification-free, OR-gated CRISPR-Cascade reaction for pathogen detection in blood samples, Proceedings of the National Academy of Sciences (2025). DOI: 10.1073/pnas.2420166122.AbstractRapid and accurate detection of DNA from disease-causing pathogens is essential for controlling the spread of infections and administering timely treatments. While traditional molecular diagnostics techniques like PCR are highly sensitive, they include nucleic acid amplification and many need to be performed in centralized laboratories, limiting their utility in point-of-care settings. Recent advances in CRISPR-based diagnostics (CRISPR-Dx) have demonstrated the potential for highly specific molecular detection, but the sensitivity is often constrained by the slow trans-cleavage activity of Cas enzymes, necessitating preamplification of target nucleic acids. In this study, we present a CRISPR-Cascade assay that overcomes these limitations by integrating a positive feedback loop that enables nucleic acid amplification-free detection of pathogenic DNA at atto-molar levels and achieves a signal-to-noise ratio greater than 1.3 within just 10 min. The versatility of the assay is demonstrated through the detection of bloodstream infection pathogens, including Methicillin-Sensitive Staphylococcus aureus (MSSA), Methicillin-Resistant Staphylococcus aureus (MRSA), Escherichia coli, and Hepatitis B Virus (HBV) spiked in whole blood samples. Additionally, we introduce a multiplexing OR-function logic gate, further enhancing the potential of the CRISPR-Cascade assay for rapid and accurate diagnostics in clinical settings. Our findings highlight the ability of the CRISPR-Cascade assay to provide highly sensitive and specific molecular detection, paving the way for advanced applications in point-of-care diagnostics and beyond.