Gene-based identification of bacterial colonies with an electric chip
2005 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 345, no 2, 270-276 p.Article in journal (Refereed) Published
A method for the identification of bacterial colonies based on their content of specific genes is presented. This method does not depend on DNA separation or DNA amplification. Bacillus cereus carrying one of the genes (hblC) coding for the enterotoxin hemolysin was identified with this method. It is based on target DNA hybridization to a capturing probe immobilized on magnetic beads, followed by enzymatic labeling and measurement of the enzyme product with a silicon-based chip. An hblC-positive colony containing 10(7) cells could be assayed in 30 min after ultrasonication and centrifugation. The importance of optimizing the ultrasonication is illustrated by analysis of cell disruption kinetics and DNA fragmentation. An early endpoint PCR analysis was used to characterize the DNA fragmentation as a function of ultrasonication time. The first minutes of sonication increased the signal due to both increased DNA release and increased DNA fragmentation. The latter is assumed to increase the signal due to improved diffusion and faster hybridization of the target DNA. Too long sonication decreased the signal, presumably due to loss of hybridization sites on the targets caused by extensive DNA fragmentation. The results form a basis for rational design of an ultrasound cell disruption system integrated with analysis on chip that will move nucleic acid-based detection through real-time analysis closer to reality.
Place, publisher, year, edition, pages
2005. Vol. 345, no 2, 270-276 p.
Bacillus cereus, enterotoxin, hemolysin, DNA fragmentation, ultrasonication, cereus diarrheal enterotoxin, bacillus-cereus, biochip technology, dna, cell, pcr, microarray, biosensors, pathogens, cytometry
IdentifiersURN: urn:nbn:se:kth:diva-15094DOI: 10.1016/j.ab.2005.07.024ISI: 000232431900010ScopusID: 2-s2.0-25444529747OAI: oai:DiVA.org:kth-15094DiVA: diva2:333135
QC 201005252010-08-052010-08-052010-08-24Bibliographically approved