Development of a sensitive DNA microarray suitable for rapid detection of Campylobacter spp.
Campylobacter is the most common cause of human acute bacterial gastroenteritis worldwide, widely distributed and isolated from human clinical samples as well as from many other different sources. To comply with the demands of consumers for food safety, there is a need for development of a rapid, sensitive and specific detection method for Campylobacter. In this study, we present the development of a novel sensitive DNA-microarray based detection method, evaluated on Campylobacter and non-Campylobacter reference strains, to detect Campylobacter directly from the faecal cloacal swabs. The DNA-microarray method consists of two steps: first, both universal bacterial sequences and specific Campylobacter sequences (size range: 149-307 bp) are amplified and fluorescently labeled using multiplex-PCR, targeting the 16S rRNA, the 16S-23S rRNA intergenic region and specific Campylobacter genes. Secondly, the Cy5 labeled PCR-amplicons are hybridised to immobilised capture probes on the microarray. The method allows detection of three to thirty genome equivalents (6-60 fg DNA) of Campylobacter within 3 h, with a hands on time of only 15 min. Using the DNA-microarrays, two closely related Campylobacter species, Campylobacter jejuni and Campylobacter coli could be detected and differentiated directly from chicken faeces. The DNA-microarray method has a high potential for automation and incorporation into a dedicated mass screening microsystem.
Keramas
Georgios
G
Mikroelektronik Centret (MIC), Technical University of Denmark, Building 345 East, Kongens 2800, Lyngby, Denmark.
Bang
Dang Duong
DD
Lund
Marianne
M
Madsen
Mogens
M
Rasmussen
Svend Erik
SE
Bunkenborg
Henrik
H
Telleman
Pieter
P
Christensen
Claus Bo Vöge
CB
D000818:Animals; D015373:Bacterial Typing Techniques / Q000379:methods; D001431:Bacteriological Techniques; D002167:Campylobacter / Q000235:genetics* / Q000302:isolation & purification*; D002169:Campylobacter Infections / Q000175:diagnosis* / Q000382:microbiology; D017000:Campylobacter coli / Q000302:isolation & purification; D016123:Campylobacter jejuni / Q000302:isolation & purification; D002645:Chickens; D005243:Feces / Q000382:microbiology*; D005516:Food Microbiology; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity