Proof-of-Concept of a Qualitative qPCR Assay for RCL Monitoring

Purpose

qPCR assays for RCL monitoring are a rapid method to detect and usually quantify lentiviral genes, such as the envelope gene vesicular stomatitis virus G glycoprotein (VSV-G). Since FDA (Food and Drug Administration) guidance requires that all RCL positive results should be pursued by direct culture assay to obtain and characterize the infectious viral isolate, we concluded, that a quantitative assay is not necessarily needed to detect RCL. Therefore, we developed an alternative method for a highly sensitive, cost-efficient qualitative qPCR assay to assess the presence of the VSV-G sequence for the purpose of RCL monitoring. Our approach is based on recommendations for qualitative anti-drug-antibody assays, where a low positive control with an error rate of 1% monitors the assay at the sensitivity level.