Rapid and sensitive point-of-care diagnosis of human cytomegalovirus infection using RPA-CRISPR technology

Abstract

Background: Human cytomegalovirus (HCMV) is a common herpesvirus that can cause a range of symptoms, from mild conditions such as fevers to severe illnesses like pneumonia. Early and accurate diagnosis of HCMV infection is crucial, particularly for vulnerable populations with limited medical care. However, current diagnostic methods are often expensive, time-consuming, and require skilled technicians. Materials and methods: We developed an HCMV-RPA-CRISPR diagnosis platform for the rapid and cost-effective detection of HCMV infection. This method utilizes recombinase polymerase amplification (RPA) to amplify the HCMV target gene isothermally without the need for thermal cycling equipment. The platform integrates the CRISPR/Cas12a system, significantly enhancing specificity and sensitivity. A total of 13 clinical blood samples were tested to evaluate the platform's effectiveness and accuracy. Additionally, a lateral flow assay (LFA) and fluorescence detection were incorporated for straightforward and rapid visual interpretation of the results. Results: The assay effectively detected concentrations as low as a single copy of the positive control plasmid per microliter in under 1 h, without requiring specialized equipment or training. In clinical sample evaluations, both the fluorescence readout and LFA exhibited 100% sensitivity and specificity, identifying four HCMV-positive and nine HCMV-negative samples. Conclusion: The HCMV-RPA-CRISPR diagnosis platform is comparably effective to qPCR for HCMV diagnosis. Its applicability in common clinical laboratories, clinics, and point-of-care settings, particularly in resource-limited environments, makes it a valuable tool for widespread HCMV screening and diagnosis.

Keywords

• Human cytomegalovirus
• HCMV
• Point-of-care diagnostics
• Recombinase polymerase amplification
• CRISPR diagnosis