B-272 Interference Assays: How Different Substances can Affect the Detection of Respiratory Viruses Using a Quick Molecular Solution

Abstract

Abstract Background Some substances present in clinical samples could affect the performance of qPCR and significantly impact its results. If nucleic acids are obtained using quick lysis reagents, the inhibition effect could be even worse since no washing steps are included. Thence, the aim of this study was to evaluate some potentially interfering substances that may be present in respiratory samples and their effect on the diagnosis using a quick molecular solution. Methods Ten negative nasopharyngeal swab samples in Viral Transport Medium (VTM, Vircell) were pooled and enriched with a known concentration of viral cultures from ATCC (Influenza A: VR-95PQ™ A/Puerto Rico/8/34; Human Respiratory Syncytial virus (RSV): VR26PQ™; and SARS-CoV-2: VR-1986HK™ SARS-Related Coronavirus 2, Isolate USA-WA1/2020) to obtain positive samples around the limit of detection. The pool was divided into seven aliquots. Six of them were spiked (each) with a known concentration of potentially respiratory interfering substances, according to FDA recommendations (Table 1). Another aliquot did not contain any substance and was considered as the positive control. Each aliquot was processed in triplicate using VIASURE Resp. viruses Quick Lysis Reagent (Certest Biotec S. L), following the manufacturer’s instructions. Nucleic acids were analysed with VIASURE SARS-CoV-2, Flu & RSV Real Time PCR Detection Kit (Certest Biotec S.L) in triplicate on CFX96™ Real-Time PCR Detection System (Bio-Rad). Results Almost all substances did not interfere throughout the workflow at the concentrations tested. Only benzocaine had a negative effect on the three pathogens analysed (SARS-CoV-2, Influenza A and RSV), detecting less than 8/9 replicates. This problem was resolved when the substance was tested at 0.15 mg/ml. Conclusion Evaluation of potentially interfering substances in quick molecular solutions is important, especially if nucleic acids processing methods do not have many purification steps. Thus, the study confirmed that the diagnosis using this solution is not easily compromised.