Optimization and Evaluation of a Novel Real-Time RT-PCR Test for Detection of Parechovirus in Cerebrospinal Fluid

Abstract Human parechovirus(HPeV) infections cause a broad array of clinical manifestations ranging from gastrointestinal or respiratory illness to central nervous system (CNS) diseases. Though nucleic acid amplification tests (NAATs) for detection of HPeVs have been described, a need exists for sensitive and specific NAATs with internal control (IC). This study describes optimization and evaluation of a novel, real-time reverse transcription PCR (RT-PCR) test for detection of HPeV from CSF using EliTech HPeV Research-Use-Only detection reagent, MS2 IC and quantified HPeV control. Four RT-PCR kits were compared to select an enzyme with optimal amplification efficiency. The optimal RT-PCR enzyme volume and the best approach to add MS2 to the easyMAG extraction platform were investigated. Following assay optimization, performance characteristics were determined. SuperScript was the most efficient one-step RT-PCR kit, with 0.5 μl/reaction of enzyme being most cost-effective. Adding MS2 to samples post-lysis was better than pre-lysis. The limit of detection of the new test was 570 copies/mL. Commercially-available HPeV 1–6 were detectable, and no cross-reactivity with other CNS pathogens was observed. This test was accurate and reproducible for detection of HPeV and IC. It demonstrated good performance characteristics and is a useful addition to a suite of molecular assays for detection of viral pathogens in CSF.