A Rapid and Sensitive Detection Cascade for <i>Bacillus subtilis</i> Vegetative Cells Utilizing Phage-Derived CBD and Lysin

<i>Bacillus subtilis</i> is a ubiquitous Gram-positive bacterium with significant industrial and agricultural applications, yet it is also associated with food spoilage and opportunistic infections. Current detection techniques are often constrained by their complexity, extended processing time, failure to differentiate viable cells, and susceptibility to interference in complex matrices. To overcome these limitations, we identified a novel lysin, proLysin, derived from phage PJNB032. Its cell-wall binding domain (CBD) contains one SH3 domain and two LysM domains and specifically targets vegetative cells of <i>B. subtilis</i>. We further demonstrated that wall teichoic acid (WTA) acts as the receptor for phage PJNB032, and that its lysin exhibits broad-spectrum lytic activity. Based on these properties, we developed an integrated cascade detection system comprising: (1) qualitative screening via fluorescently labeled CBD, achievable within 10 min; (2) quantitative immunomagnetic capture completed within 30 min; and (3) enzymatic specificity validation through proLysin-mediated lysis, enabling target identification within 30 min. This method exhibits a detection limit of 9 CFU/mL for vegetative <i>B. subtilis</i> cells and demonstrates high accuracy across challenging sample types such as soil, forage, and water. This study establishes a rapid, sensitive, and reliable platform for the detection of viable <i>B. subtilis</i>, offering a superior alternative to conventional methods for quality control in its fermentation processes and environmental monitoring.