Evaluation of the performance of reverse transcription-recombinase polymerase amplification (RT-RPA) coupled with CRISPR/Cas12a and microfluidics for one-step detection of common HCV genotypes.

BACKGROUND

Hepatitis C virus (HCV) genotyping is critical for guiding therapy, yet current methods are technically demanding and time-consuming.

AIM: To develop and evaluate a one-step, integrated assay combining reverse transcription-recombinase polymerase amplification (RT-RPA) with CRISPR/Cas12a detection on a microfluidic platform for rapid and accurate HCV genotyping.

METHODS

The assay was validated using 186 clinical samples genotyped by Sanger sequencing. The microfluidic chip enabled sequential RT-RPA amplification and CRISPR/Cas12a detection via centrifugal fluid transfer, with real-time fluorescence monitoring.

RESULTS

The assay demonstrated high concordance with Sanger sequencing (overall accuracy >98%), with sensitivities of 100% for genotypes 1b and 6a, and >97% for 2a and 3a. The limit of detection was 1 IU/mL (5 copies/mL)across major genotypes, with no cross-reactivity against other viruses.

CONCLUSION

The integrated RT-RPA-CRISPR/Cas12a-microfluidics platform offers a rapid, sensitive, and specific one-step assay for HCV genotyping, suitable for point-of-care applications in resource-limited settings.