Abstract

Helicoverpa armigera nucleopolyhedrovirus (HearNPV) is a biological control agent used for the management of the cotton bollworm Helicoverpa armigera, an insect pest that causes significant damage to a wide range of economically important crops. The development and use of biological pesticides containing HearNPV have contributed to reducing the adverse impacts of chemical pesticides and are consistent with the orientation toward sustainable agricultural development. However, the conventional method for quantifying viral occlusion bodies (OBs) using a hemocytometer has several limitations in terms of accuracy, repeatability, and the ability to distinguish OBs from particulate excipients present in the formulation matrix. Real-time PCR enables the sensitive, specific, and highly reproducible detection and quantification of viral DNA and has been successfully applied for the quality control of various viral preparations worldwide.

In this study, a real-time PCR method for the quantification of HearNPV occlusion bodies was developed based on the amplification of a virus-specific fragment of the DNA polymerase gene. DNA extracted from samples with known concentrations, determined using a hemocytometer, was used to establish a standard curve, which served as the basis for quantifying the corresponding OB concentration in plant protection products. The results showed that the developed method achieved 100% accuracy, sensitivity, and specificity, with a limit of detection and limit of quantification of 102 OBs/mL, while no amplification signal was observed in the negative control samples. The standard curve demonstrated a strong linear correlation between the Ct values and log10 viral OB concentration (R² = 0.995), with an amplification efficiency of 90.5%. Both repeatability and reproducibility met the acceptance criteria specified in ISO 22118:2011. These findings demonstrate the potential of the developed method for the quantification and quality control of plant protection products containing HearNPV.

Keywords:

Helicoverpa armigera nucleopolyhedrovirus; real-time PCR; virus quantification; plant protection products; hemocytometer.

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