AUTHOR=Miao Zhuohan , De Buck Jeroen TITLE=Discriminating bovine mastitis pathogens by combining loop-mediated isothermal amplification and amplicon-binding split trehalase assay JOURNAL=Frontiers in Veterinary Science VOLUME=11 YEAR=2024 URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2024.1389184 DOI=10.3389/fvets.2024.1389184 ISSN=2297-1769 ABSTRACT=

Bovine mastitis is predominantly caused by intramammary infections with various Gram-positive and Gram-negative bacteria, requiring accurate pathogen identification for effective treatment and antimicrobial resistance prevention. Here, a novel diagnostic method was developed to detect mastitis pathogens in milk samples by combining loop-mediated isothermal amplification with a split enzyme biosensor whereby trehalase fragments were fused with a DNA-binding protein, SpoIIID. Three primer sets, LAMPstaph, LAMPstrep, and LAMPneg, harboring SpoIIID recognition sequences targeted Staphylococcus, Streptococcus, and Gram-negative pathogens, respectively. Limits of detection were determined for DNA extracted from bacterial culture and bacteria-spiked milk. The combined method detected as low as 2, 24, and 10 copies of genomic DNA of staphylococci, streptococci and Escherichia coli and 11 CFU/ml for milk spiked with Escherichia coli. Higher detection limits were observed for Gram-positive bacteria in spiked milk. When testing genomic DNA of 10 mastitis isolates at concentrations of 106 and 103 copies per reaction, no cross-reactivity was detected for LAMPstaph nor LAMPstrep, whereas the LAMPneg assay cross-reacted only with Corynebacterium sp. at the highest concentration. This combined method demonstrated the potential to distinguish mastitis pathogenic Gram types for a rapid decision of antimicrobial treatment without culturing.