Development and validation of a qPCR method for quantification of Spodoptera exigua nucleopolyhedrovirus in plant protection products

Abstract

Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) is an important biological control agent widely used as an eco-friendly alternative to chemical pesticides for managing Spodoptera exigua. The efficacy of SeMNPV-based plant protection products strongly depends on the concentration of viral occlusion bodies (OBs); however, conventional quantification methods based on hemocytometer counting are labor-intensive, time-consuming, and subject to operator bias. Therefore, this study aimed to develop and validate a real-time PCR (qPCR) method for the quantification of SeMNPV in plant protection products. In this study, a qPCRbased method for the quantification of SeMNPV was developed by targeting amplification of the dnapol gene, which represents a highly conserved and species-specific genetic marker for this virus. Viral identity was confirmed by dnapol gene sequencing, and hemocytometer-based quantification of viral OBs was employed to determine reference input values for qPCR validation. Method validation was conducted in accordance with ISO 22118:2011 guidelines, evaluating analytical sensitivity, specificity, accuracy, linearity, repeatability, and reproducibility. The assay demonstrated high sensitivity, with both the limit of detection and the limit of quantification established at 10² OBs/mL. Specificity, accuracy, and sensitivity reached 100%, with no crossreactivity observed against non-target baculoviruses. A robust standard curve based on the linear relationship between Ct values and OB concentration showed excellent linearity (R² = 0.994), a slope of -3.151, and an amplification efficiency of 107.6%. Repeatability and reproducibility met acceptance criteria, indicating good precision and method stability. Overall, this study presents the first validated qPCR method for SeMNPV quantification, validated against occlusion body counts, providing a sensitive, specific, and biologically relevant tool for quality control and regulatory assessment of SeMNPV-based biopesticides.