Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10429520 | Biosensors and Bioelectronics | 2011 | 7 Pages |
Abstract
Members of the genus Cronobacter are opportunistic pathogens formerly known as Enterobacter sakazakii, which induce severe meningitis and sepsis in neonates and infants, with a high fatality rate. In this work, a simple and rapid immunochromatographic strip test for the detection of this pathogen was developed. Following the shortened bacteria cultivation and isolation of DNA, a specific gene sequence targeting 16S rRNA from Cronobacter spp. was amplified by PCR using 5â²-end labelled specific primers. The PCR product, amplicon labelled with digoxigenin on one side and biotin on the other side, was directly added to the immunochromatographic strip test, composed of nitrocellulose membrane with bound antibody against digoxigenin in the test line. The visualization was mediated by colloidal carbon conjugated to neutravidin, and the appearance of grey/black line was indicative of the presence of specific amplicon. Colour intensity of the test line in pathogen-positive assay was visually distinguishable from that of negative sample within 10Â min. The visual detection limit of PCR product was 8Â ng. The specificity of the developed method was confirmed by standard microbiological techniques. Whole detection procedure with the incorporated immunostrip was applied to analysis of infant formulae samples, contaminated with less than 10 cells of Cronobacter spp. per 10Â g. The results from immunochromatographic test indicated the absolute agreement with those from standard microbiological methods. Moreover, the developed procedure considerably reduced the total analysis time to 16Â h whereas the reference microbiological method needs 6-7 days.
Keywords
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Martina Blažková, Barbora Javůrková, Ladislav Fukal, Pavel Rauch,