A novel generic dipstick-based technology for rapid and precise detection of tetracycline, streptogramin and...

Excessive use of antibiotics in veterinary medicine and as growth promoters in stock farming has been associated with the dramatically increasing prevalence of multidrug-resistant human pathogenic bacteria. European community legislators have therefore restricted the veterinary use of antibiotics and banned them as growth-promoting food additives in stock breeding (1831/2003/EC). The monitoring of such legislation requires technology for precise and straightforward on-site quantification of antibiotics in farm samples and food products without the need for extensive laboratory equipment and trained personnel. Capitalizing on bacterial transcriptional regulators (TetR, PIP, E), which are dose-dependently released from their cognate operators (tetO, PIR, ETR) upon binding of specific classes of antibiotics (tetracycline, streptogramins, macrolides) we have designed an easy-to-handle dipstick-based assay for detection of antibiotic levels in serum, meat and milk whose detection limits are up to 40-fold below licensed threshold values. The generic dipstick consists of either nitrocellulose, nylon or polyvinylidenfluorid (PVDF) membrane strips coated with streptavidin and immobilized biotinylated operator DNA, which acts as capture DNA to bind hexa-histidine (His(6))-tagged bacterial biosensors. Antibiotics present in specific samples triggered the dose-dependent release of the capture DNA-biosensor interaction, which, after dipping into two different solutions, results in a correlated conversion of a chromogenic substrate by a standard His(6)-targeted enzyme complex. This can be quantified by comparison of the dipstick to a standardized color scale or by assessing the terminal solution at 450nm. As demonstrated using serum, meat and milk samples spiked with 14 different antibiotics, the dipstick technology provided sensitive detection in a rapid assay format, and could be employed to monitor non-authorized use of antibiotics and to discover novel antibiotics.