[Quantification of Influenza A/B Virus and RSV Validation Standards using Digital PCR]

Method verification or validation studies using quantitative standards are required for diagnostic tests to be used in microbiological diagnostic laboratories. Standards containing whole viruses instead of synthetic nucleic acids or plasmids ensures that all steps of the assay, including extraction, reverse transcription and polymerase chain reaction (PCR), are evaluated as in real life. Commercial quantitative standard materials for nucleic acid tests are limited for respiratory viruses. In this study, we aimed to develop quantitative nucleic acid standards for influenza A (infA), influenza B (infB) viruses and respiratory syncytial virus (RSV) using digital PCR (dPCR). In the study, the amount of viral nucleic acids in samples prepared by pooling nasopharyngeal swab samples known to be positive for RSV, infA, infB RNA were determined by dPCR (QIAcuity, Qiagen) using commercial primer/probe sets (Qiagen, Germany). Nucleic acid extraction was performed using a commercial kit (Xi’an Tianlong Science&Technology Co, China). The analytical sensitivity (LoD) and lower limit of quantitation (LoQ), intra- and inter-study reproducibility and linearity of the dPCR method for infA, infB and RSV were determined. Samples were analysed by both dPCR and real-time RT-PCR (qRT-PCR) and the relationship between Ct values and dPCR quantification results was evaluated by linear regression. Statistical analysis were performed using GraphPad Prism 10.4.0 (GraphPad, USA) and Excel Analysis ToolPak. The LoD values of the dPCR method for infA, infB and RSV were 93.75, 15.59 and 26.23 copies/mL, respectively. The intra-study reproducibility (coefficient of variation, CV%) of the dPCR method ranged from 0.06 to 7.97, being higher in samples with low viral load. Inter-study reproducibility was 0.73 to 5.41. Linearity analysis performed with dilutions in the range of 3-4 log10 for infA and in infB and seven log10 for RSV showed r2≥ 0.99 for all three viruses. The concentrations measured by dPCR were correlated with qRT-PCR Ct results. When the intra- and inter-study reproducibility results of dPCR and qRT-PCR tests were compared, the CV% value of dPCR was significantly lower (p= 0.0312). The results of the study showed that the dPCR method is a highly reproducible and reliable method for obtaining quantitative standards. The quantitative standards obtained can be used to develop assays for viral load determination and/or to perform method confirmation analysis of such assays. In conclusion, in this study reliable quantitative nucleic acid standards for infA, infB and RSV were obtained by dPCR using pooled patient samples and performance analysis of the dPCR method was determined. This study was an example of the production of quantitative viral nucleic acid standards by dPCR.