Direct On-Chip Diagnostics of Streptococcus bovis/Streptococcus equinus Complex in Bovine Mastitis Using Bioinformatics-Driven Portable qPCR

This study introduces an innovative on-site diagnostic method for rapidly detecting the <i>Streptococcus bovis</i>/<i>Streptococcus equinus</i> complex (SBSEC), crucial for livestock health and food safety. Through a comprehensive genomic analysis of 206 genomes, this study identified genetic markers that improved classification and addressed misclassifications, particularly in genomes labeled <i>S. equinus</i> and <i>S. lutetiensis</i>. These markers were integrated into a portable quantitative polymerase chain reaction (qPCR) that can detect SBSEC species with high sensitivity (down to 10¹ or 10⁰ colony-forming units/mL). The portable system featuring a flat chip and compact equipment allows immediate diagnosis within 30 min. The diagnostic method was validated in field conditions directly from cattle udders, farm environments, and dairy products. Among the 100 samples, 51 tested positive for bacteria associated with mastitis. The performance of this portable qPCR was comparable to laboratory methods, offering a reliable alternative to whole-genome sequencing for early detection in clinical, agricultural, and environmental settings.