Development of cloth-based hybridization systems for the detection and characterization of foodborne pathogenic bacteria

A cloth-based hybridization array system (CHAS) approach was developed in which a multiplex polymerase chain reaction (PCR) incorporating digoxigenin-dUTP was used for simultaneous amplification of multiple target sequences, with subsequent rapid detection of the amplicons by hybridization with an array of probes immobilized on polyester cloth, and immunoenzymatic assay of the bound digoxigenin label. Three separate CHAS were developed: (1) a CHAS for detection of multiple antibiotic resistance and other marker genes in the characterization of Salmonella enterica subsp. enterica serotype Typhimurium DT104 isolates; (2) a CHAS for the detection of various toxin genes associated with major foodborne pathogenic bacteria; and (3) a CHAS for the detection and identification of different Clostridium botulinum toxin gene types. In addition, a cloth-based dot blot hybridization system (C-DBHS) was developed in which the PCR products are assayed by spotting on a polyester cloth sheet capable of accommodating multiple samples, with subsequent detection of the amplicons by hybridization with a digoxigenin-labelled target-specific DNA probe and immunoenzymatic assay of the bound label.