Molecular Characterization of Callosobruchus maculatus (Fabricius, 1775), DNA Barcoding and Development of Multiplex PCR for Detection of Stored Grain Pests

Abstract

The present study aimed to characterize Callosobruchus maculatus populations collected from five distinct locations in Andhra Pradesh, India (Tirupati, LAM, Guntur, Krishna and Nandyal), utilizing mitochondrial cytochrome oxidase I (mtCO1) gene-based DNA barcoding. Additionally, the research sought to develop a molecular diagnostic tool for detecting hidden infestations in stored pulses. PCR amplification employing universal primers yielded fragments of approximately 750 bp. Subsequent nBLAST analysis confirmed these sequences exhibited >99% similarity with C. maculatus entries in GenBank. The generated sequences were submitted to the National Center for Biotechnology Information (NCBI), and unique DNA barcodes were registered in the Barcode of Life Data (BOLD) systems. Notably, this represents the first documented report of C. maculatus DNA barcodes from Andhra Pradesh. Phylogenetic analysis indicated low intraspecific divergence (<1%) among the examined C. maculatus populations from Andhra Pradesh, with populations from Krishna and Tirupati demonstrating complete sequence identity. Furthermore, a multiplex PCR assay targeting the mtCO1 gene was developed to enable simultaneous detection of C. maculatus, Sitophilus oryzae and Rhyzopertha dominica. This multiplex PCR produced distinct amplicons of 119 bp, 95 bp and 143 bp for the respective species, proving to be a rapid, sensitive, and cost-effective method for identifying hidden infestations in pulses. The findings underscore the efficacy of DNA barcoding for genetic characterization of pulse beetle populations and highlight the potential of multiplex PCR as a robust, high-throughput diagnostic tool for managing stored grain pests.