A Simple, Rapid, and Contamination-Free Ultra-Sensitive Cronobacter sakazakii Visual Diagnostic Platform Based on RPA...

Area of Science:

• Molecular Diagnostics
• Foodborne Pathogen Detection
• CRISPR/Cas Systems

Background:

• CRISPR/Cas systems offer significant advancements in molecular diagnostics.
• The common two-step method for nucleic acid detection faces challenges with aerosol contamination.
• One-pot methods aim to simplify detection but often require specialized equipment or modifications.

Purpose of the Study:

• To develop a novel one-pot strategy for detecting the foodborne pathogen Cronobacter sakazakii (C. sakazakii).
• To establish a rapid detection platform for C. sakazakii using CRISPR/Cas12a and recombinase polymerase amplification (RPA).
• To address aerosol contamination issues in RPA-CRISPR/Cas12a methods and develop a simplified, sensitive detection strategy.

Main Methods:

• A specific sequence from the C. sakazakii virulence gene ompA was identified as the detection target.
• A CRISPR/Cas12a system was combined with RPA to create a rapid detection platform.
• A novel pipette tip-in-tube (PTIT) method was developed to isolate RPA and CRISPR/Cas components, preventing aerosol contamination in a one-pot format.

Main Results:

• The developed method demonstrated high sensitivity, with detection limits of 10⁻⁴ ng/μL for genomic DNA, 1.43 copies/μL for target DNA, and 6 CFU/mL for pure bacterial culture.
• Detection limits in artificially contaminated milk powder samples were as low as 4.65 CFU/mL (cow milk) and 4.35 CFU/mL (goat milk) without enrichment.
• The PTIT method achieved the same sensitivity as the two-step method but completely eliminated false positives due to aerosol contamination, without requiring special devices or chemical modifications.

Conclusions:

• A novel one-pot CRISPR/Cas12a detection strategy, the PTIT method, was successfully developed for sensitive and rapid detection of C. sakazakii.
• The PTIT method effectively overcomes aerosol contamination issues inherent in traditional methods, offering a simplified and robust solution.
• This approach holds significant potential for rapid point-of-care testing (POCT) applications due to its ease of use and high sensitivity.