The RPA-CRISPR/Cas12a system in this study achieves rapid (30–40 min), ultra-sensitive (1 copy/μL) HCMV detection by targeting the early-expressed UL123 gene, outperforming conventional methods in clinical sample testing.This innovation enables timely diagnosis of low-viral-load infections, particularly in immunocompromised patients, and underscores the feasibility of CRISPR-based diagnostics for improving management of HCMV-related diseases. The assay's simplicity and efficiency highlight its potential as a point-of-care tool for resource-limited settings.
Patterns of RPA amplification sites and kit selection.
(A) RPA primer design regarding the location of amplification fragments. Arrows indicate the base positions from the beginning to the end of the amplified fragment with the P1-P6 primers.
(B) The agarose gel electrophoresis image of the RPA product amplified with 6 HCMV primer sets.
(C) Screening of the amplification efficiency of RPA kits from different companies. P: Primer, NC: Negative Control.
The results indicated that the AmpFuture kit exhibited the highest amplification efficiency.
Comparison of positive rates of MIRA-CRISPR/Cas12a, PCR - Fluorescent Probe Method and qPCR in clinical samples.
We collected blood and urine samples from 48 clinical patients. Genomic DNA was extracted from these samples using the TIANamp Genomic DNA Kit, and the results of UL122 detection were analyzed via the PCR-fluorescent probe method. Comparing our results with clinical diagnoses, our methods identified 6 positive samples that were negative in clinical tests.
