Enterovirus Testing in Hand, Foot, and Mouth Disease and Herpangina: A Highly Sensitive Single-Round VP4–VP2 Reverse-Transcription Polymerase Chain Reaction Assay with a Redesigned Reverse Primer

Molecular typing of enteroviruses (EVs) is essential for surveillance of hand, foot, and mouth disease (HFMD). Conventional reverse-transcription polymerase chain reaction (RT-PCR) targeting the VP4–VP2 region can be insufficiently sensitive, reducing the detectability of Enterovirus A (EV-A). We developed a single-round RT-PCR assay using a modified reverse primer design (C3R) for rapid EV detection and genotyping. Sensitivity was evaluated using EV-A71 and poliovirus type 1 reference strains and 60 EV-positive clinical specimens. The C3R-based assay showed ~1,000-fold higher sensitivity for EV-A71 than conventional assays (limit of detection: 6.6 copies/reaction). It detected 100% (60/60) of clinical specimens, whereas the conventional assay detected only 45.0% (27/60) and failed at cycle threshold (Ct) values >30. Our assay maintained 100% sensitivity even at low viral loads (Ct > 35; P < 0.0001). All amplicons yielded high-quality sequences for definitive genotyping. This C3R-based RT-PCR overcomes sensitivity limitations of existing protocols and provides reliable genotyping from low-copy specimens, supporting its use in routine diagnostics and large-scale HFMD surveillance.