Evaluation of various filter paper and reagent systems for the preservation of Newcastle disease virus RNA samples.

Introduction: The transport of Newcastle disease virus (NDV) specimens, isolates or purified RNA is traditionally performed at ultra-low temperatures using dry ice to prevent degradation. However, this method is costly and requires specialised packaging and stringent shipping conditions. The aim of this study is to evaluate existing products' capacities to preserve NDV or its RNA under different conditions. Material and Methods: Flinders Technology Associates (FTA) cards, RNASound cards, and RNAstable tubes were tested for their ability to preserve NDV RNA at ambient temperatures. Two controls - free RNA and free virus - were included for comparison. Preservation was evaluated at various storage conditions (-80°C, -20°C, 4°C, 25°C and 56°C) and incubation times (1, 7, 14, 28 and 35 d) using a reverse-transcription PCR, Sanger sequencing and ratiometric fluorometry. Results: All preservation methods performed effectively at lower temperatures. The FTA cards maintained consistent RNA integrity with Δ threshold cycles < 2 except at 56°C on days 14-35. RNASound preserved RNA stably but was inconsistent on day 35 at 56°C. RNAstable was effective at intermediate times but had allowed complete degradation by day 35. Free RNA degraded rapidly after day 1, while free virus initially remained stable but deteriorated over time. Sanger sequencing confirmed high-quality recovery, except for recovery of free RNA, which lacked long-term stability. Conclusion: Despite challenges with prolonged storage and high temperatures, these methods demonstrated satisfactory performance. They offer viable alternatives to ultra-low temperature storage, enabling sample transport at ambient temperatures while preserving RNA integrity, and could be particularly useful in remote settings.