Optimization of a multiplex RT-PCR assay for the detection of human parechovirus, enterovirus, adenovirus type 14, and respiratory syncytial virus using the panther fusion system.

Influenza-like illnesses (ILI) are a significant cause of morbidity and mortality for children, elderly, and immunocompromised individuals. Several ILIs are caused by viruses of clinical and epidemiological importance, including human parechovirus (HPeV), human enterovirus (EV), human adenovirus 14 (HAdV14), and respiratory syncytial virus (RSV). The Panther Fusion System provides the capability for laboratories to perform fully automated nucleic acid extraction and RT-PCR through design and optimization of a laboratory developed test (LDT) using their Open Access feature. This study reports the optimization of a real time reverse transcription polymerase chain reaction (RT-PCR) LDT for HPeV, EV, HAdV14, and RSV using the Panther Fusion System. The primer probe reconstitution mix (PPR) concentrations were optimized to 0.6 µM primers, 0.4 µM probes, 3 mM MgCl2, and 70 mM KCl. The onboard stability of the PPR was determined to be 14 days. Accuracy was assessed using nasopharyngeal, oropharyngeal, and nasal swab specimens spiked with HPeV, EV, HAdV14, and RSV positive culture fluids, with 100 % detection for all targets. Cerebrospinal fluid spiked with EV-D68 and HPeV3 was also assessed for accuracy, with 100 % detection for both EV-D68 and HPeV3. The limit of detection for this assay was 2 × 102 copies/µl for all targets. This study demonstrates the successful optimization of a multiplex RT-PCR LDT for the detection of HPeV, EV, HAdV14, and RSV.