Researchers have developed a gelatin-assisted recombinase polymerase amplification and CRISPR/Cas12a tandem assay to detect Listeria monocytogenes with high sensitivity. Combining isothermal nucleic acid amplification with the CRISPR/Cas12a platform offers significant analytical specificity, and the newly reported method introduces a physical separation mechanism to enhance performance.

The assay, termed GARC, relies on temperature-responsive gelatin to physically encapsulate CRISPR/Cas12a reagents. During recombinase polymerase amplification incubation at 37 °C, the gelatin remains in a solid gelled state, preventing premature interaction between the amplification machinery and CRISPR components.

In addition to preserving reaction integrity during isothermal incubation, gelatin at an appropriate concentration enhances fluorescence signal intensity, facilitating a direct visual readout of the reaction outcome.

In analytical sensitivity trials, the optimized GARC system achieved a limit of detection of 10 ag/μL for the hlyA target gene. This analytical threshold represents a 10,000-fold improvement over paraffin-based one-pot RPA-CRISPR methods, which exhibited a limit of detection of 100 fg/μL.

The method also outperformed conventional polymerase chain reaction protocols by a factor of 10,000,000, where standard PCR achieved a detection threshold of 100 pg/μL. When evaluating bacterial cells, the platform successfully identified 8 CFU/mL of Listeria monocytogenes.

The assay demonstrated detection capabilities down to 1.03 × 10−5 ng/μL of bacterial genomic DNA. Across specificity and sensitivity assessments, the testing platform achieved favorable scores of 5/5 for sensitivity and 16/16 for analytical specificity.

Practical applicability was evaluated using contaminated food matrices, specifically salmon samples. The platform successfully detected the pathogen at an inoculation level of 20 CFU per 25 grams of salmon following a 7-hour enrichment process.

This work introduces a versatile, visually interpretable platform with potential for food safety testing.

In evaluating analytical limits against standard genetic targets, the testing system pinpointed Listeria monocytogenes through detection of the hlyA gene.

Subject of Research: Rapid detection of Listeria monocytogenes using a gelatin-assisted RPA-CRISPR/Cas12a tandem assay

Article Title: Gelatin assisted RPA-CRISPR/Cas12a tandem assay for rapid detection of Listeria monocytogenes

Article References: Du, W., Wu, S., Wu, M., Zhao, K., Chen, Y., Sha, X., Shao, Y., Chen, X., Lai, W., Wang, S., Liu, D., & Shan, S. (2026). Gelatin assisted RPA-CRISPR/Cas12a tandem assay for rapid detection of Listeria monocytogenes. npj Science of Food. https://doi.org/10.1038/s41538-026-01211-w

Image Credits: AI Generated

DOI: 10.1038/s41538-026-01211-w

Keywords: Listeria monocytogenes, CRISPR-Cas12a, recombinase polymerase amplification, GARC assay, food safety testing, isothermal amplification, pathogen detection, bacterial diagnostics, gelatin encapsulation, hlyA gene, fluorescence readout, salmon testing, analytical sensitivity, limit of detection, foodborne pathogens, molecular diagnostics