Abstract

Xanthomonas oryzae pv. oryzae (Xoo) is a significant pathogen responsible for seed-borne diseases that negatively impact rice yields globally. Effective early detection methods for this pathogen, particularly those based on nucleic acid detection, are crucial for maintaining plant health. This study presented an electrochemical DNA biosensor that utilized a novel pyrrolidinyl peptide nucleic acid (acpcPNA) probe sequence derived from the wxoC gene, which encoded a putative glycosyltransferase, for sensitive, selective, and direct detection of Xoo in rice seed samples. The acpcPNA probe was designed, synthesized, and characterized using mass spectrometry and melting temperature analysis. It was then immobilized onto a graphene oxide-bovine serum albumin (GO/BSA) nanocomposite modified electrode to demonstrate its electrochemical sensing capabilities, as GO/BSA aided in stabilizing film formation, leading to an effective immobilization process. The formation of the probe with Xoo double-stranded DNA (dsDNA) via duplex invasion considerably reduced the redox signal response due to electrostatic repulsion and insulating properties. With the optimized conditions, the sensor demonstrated a linear detection range from 10 to 20 × 10³ nM for synthetic DNA and 10³ to 10⁸ CFU/mL for bacterial cell culture, with detection and quantification limits of 1.8 and 10 nM, and 1 × 10³ and 3.3 × 10³ CFU/mL, respectively. This biosensor successfully detected Xoo dsDNA in artificially and naturally infected rice seeds without pre-enrichment and denaturation in a label-free format within 45 min, as verified by polymerase chain reaction. Furthermore, the system offered exceptional specificity, making it a promising tool for the early detection of this pathogen.