Rapid detection of mutans streptococci by substrate specific binding of automutanolysin : Final report

Chair-side rapid detection of mutans streptococci is an important aid to clinical dental caries risk assessment. Rapid Streptococcus mutans detection tools are available on the market but there are a small number. Automutanolysin (Aml) is a peptidoglycan hydrolase whose cell wall-binding domain (CWBD) has substrate-specificity towards mutans streptococci. This study aims to develop a rapid detection assay using CWBD conjugated with horseradish peroxidase (HRP). However, the recombinant protein was as insoluble form. Therefore, magnetic nanoparticles were used as an alternative reporter to conjugate with CWBD (CWBD-conjugated MNPs). Magnetic nanoparticles were grafted with poly(acrylic acid) (PAA) providing active carboxyl groups for conjugation with CWBD of Aml. To determine binding ability and specificity of CWBD-conjugated MNPs against mutans streptococci and Streptococcus sanguinis and Streptococcus salivarius, the composite particles were mixed with single-species bacterial solution and then isolated with external magnet. The isolated CWBD-conjugated MNPs were then re-suspended in phosphate buffer saline. Bacteria-bound CWBD-conjugated MNPs were separated from unbound one by vacuum filtration through a 0.8 µm nitrocellulose filter membrane. There was a linear relationship between the intensity of colored spots of filtered-MNPs on a membrane and the number of mutans streptococci (range 10²-10⁷ CFU/ml) but not with other two streptococci. Addition of 3,3’,5,5’-Tetramethylbenzidine (TMB) and hydrogen peroxide (H₂O₂) enhance a signal of MNPs and thus improve the detection limit. For clinical application, the detection ability against mutans streptococci in mixed culture and also patient’s saliva will be further examined.