Evaluation of Tissue Direct PCR for Simplified DNA Preparation and Molecular Identification of Spodoptera frugiperda across Life Stages

Abstract

Aim: The study aimed to evaluate the Tissue Direct PCR method as a simplified and time-efficient approach for DNA preparation from different developmental stages of Spodoptera frugiperda and to assess its suitability for molecular identification. Methodology: A total of 13 biological samples representing eggs, larvae, pupae, female adults and male adults were collected from a laboratory-maintained population of S. frugiperda. Samples were processed using a Tissue Direct PCR kit, and DNA quality and quantity were assessed spectrophotometrically. The extracted DNA was used for amplification of the mitochondrial cytochrome oxidase I (MtCOI) barcode region. Selected PCR products were subjected to Sanger sequencing, and sequences were compared with reference sequences in the NCBI database using BLASTn. Results: Tissue Direct PCR successfully produced DNA suitable for PCR amplification across all developmental stages. The DNA concentration ranged from 528 to 1140 ng/µL, and amplification of the MtCOI region produced an approximately 700 bp product in all 13 samples. Seven representative PCR products were selected for sequencing, and the resulting sequences showed 100% similarity with S. frugiperda reference sequences in the NCBI BLASTn database, confirming species identity. The Tissue Direct PCR method also produced significantly higher DNA concentration and spectrophotometric purity parameters than the CTAB method in the 13 matched samples evaluated. Interpretation: Tissue Direct PCR simplified DNA preparation by reducing conventional extraction steps and generated DNA suitable for PCR and sequencing-based identification across developmental stages of S. frugiperda. The method shows potential for rapid molecular identification and DNA barcoding applications; however, validation using larger sample sets, additional field populations and closely related Spodoptera species is required before its use in large-scale diagnostic screening.