Impact of RNA Degradation on Viral Diagnosis: An Understated but Essential Step for the Successful Establishment of a Diagnosis Network

Abstract

The current global conditions, which include intensive globalization, climate changes, and viral evolution among other factors, have led to an increased emergence of viruses and new viral diseases; RNA viruses are key drivers of this evolution. Laboratory networks that are linked to central reference laboratories are required to conduct both active and passive environmental surveillance of this complicated global viral environment. These tasks require a continuous exchange of strains or field samples between different diagnostic laboratories. The shipment of these samples on dry ice represents both a biological hazard and a general health risk. Moreover, the requirement to ship on dry ice could be hampered by high costs, particularly in underdeveloped countries or regions located far from each other. To solve these issues, the shipment of RNA isolated from viral suspensions or directly from field samples could be a useful way to share viral genetic material. However, extracted RNA stored in aqueous solutions, even at −70 °C, is highly prone to degradation. The current study evaluated different RNA storage conditions for safety and feasibility for future use in molecular diagnostics. The in vitro RNA-transcripts obtained from an inactivated highly pathogenic avian influenza (HPAI) H5N1 virus was used as a model. The role of secondary structures in the protection of the RNA was also explored. Of the conditions evaluated, the dry pellet matrix was best able to protect viral RNA under extreme storage conditions. This method is safe, cost-effective and assures the integrity of RNA samples for reliable molecular diagnosis. This study aligns with the globally significant “Global One Health” paradigm, especially with respect to the diagnosis of emerging diseases that require confirmation by reference laboratories.

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools

“…Additionally, a very strong correlation was observed between the number of viral reads generated through sequencing and the starting number of viral copies, indicating sequencing reads can be predicted by viral copies in a sample and vice versa, which has been confirmed by other studies as well [28]. Interestingly, the observation that the sensitivity of sequencing was higher from cell culture virus spiked into serum as opposed to clinical serum samples suggests that sample handling or perhaps the quality of the sample was an important factor for sequencing sensitivity [79], thus emphasizing the importance of careful handling, transporting, and storing of clinical samples to protect the viral RNA from degradation [80,81]. This also suggests that on-site sequencing of samples as opposed to a centralized diagnostic system may allow for higher sensitivity of detection due to the ability to immediately process samples after sampling.…”

Stability of SARS-CoV-2 in Phosphate-Buffered Saline for Molecular Detection

“…Here, we show that the stability of SARS-CoV-2 can be maintained at 4°C for up to a month when –80°C storage is not available ( 6 , 7 ). At viral loads of >5,000 copies/ml—corresponding to >75% of positive samples recovered in our clinical lab to date—different storage temperatures did not have a substantial impact on our ability to detect SARS-CoV-2 when stored in PBS.…”

The impact of viral transport media on PCR assay results for the detection of nucleic acid from SARS-CoV-2 and other viruses

“…With a concentration of RNA that gave a Ct value of approximately 21 in both PBS and VTM-1, this represents a reduction of approximately ten thousand-fold and cannot be ignored. While it might be argued that the adverse impact on whole virus appeared to be slight, free nucleic acid and perhaps whole virus was destroyed at levels that could be of diagnostic relevance (20). While the impact on RNA viral genomes is likely to be markedly greater than on DNA sequences, the outcome cannot be predicted as secondary structure may also have an influence (20) and the speed and severity of the impact may vary depending on the nucleic acid target, as shown by the differences between the results for the two RNA viruses and the XIPC.…”