Development of a real-time PCR method for detection and enumeration of Plutella xylostella granulovirus in plant protection product

Tóm tắt

Plutella xylostella granulovirus (PlxyGV) is a baculovirus that specifically infects diamondback moths (Plutella xylostella) and is widely used in commercial biopesticide formulations. To ensure the quality and efficacy of PlxyGV-based biopesticides, this study developed a quantitative real-time PCR (qPCR) method for viral DNA detection and quantification. A 222 bp fragment of the ORF30 gene encoding the envelope protein ODV-E66 was cloned into the pGEM-T Easy vector, transformed into E. coli DH5α, and verified by Sanger sequencing, showing ≥ 99% identity with the published ORF30 sequence of PlxyGV (GenBank accession No. MN099286.1). The real-time PCR method demonstrates high sensitivity, with both the limit of detection and limit of quantification reaching 10¹ copies/µL. The assay exhibits specificity and accuracy are 100%. A standard curve was established showing a strong linear correlation between cycle values and the log of viral DNA concentration (R2 = 0.9957), with an amplification efficiency of 90.1%. Repeatability (RSDr = 0.20%) and reproducibility (RSDR = 0.28%) were within acceptable ranges based on ISO 22118:2011, confirming the method's reliability and applicability for quantifying PlxyGV in plant protection products.