High-speed gel microelectrophoresis, a new and easy approach for detection of PCR-amplified microbial DNA from environmental and clinical samples in microgels using conventional equipment.

AIMS: Microelectrophoresis allows the detection of DNA bands using minimal amounts of sample in a short time, but commonly requires the use of special equipment which is not available in all laboratories. This fact has limited the application of this technique in microbiology despite its advantages. In this work, we describe a new approach to perform gel microelectrophoresis, named high-speed gel microelectrophoresis (HSGME), and its application for rapid detection of bacteria, protozoa and viruses in clinical, vegetal and environmental samples. METHODS AND RESULTS: Aliquots of 0.4-1 microl of PCR product were loaded in 2 cm 1% agarose microgels and electrophoresed at high voltage (125 V cm(-1)) in conventional submarine horizontal mini-slabs. By using HSGME, single-DNA bands obtained after specific-PCR useful in diagnosis of different diseases caused by micro-organisms were detected in 5 min. CONCLUSIONS: HSGME is a rapid and easy procedure applicable to detection of microbial genes, which is carried out using conventional equipment and thus can be performed in any research and diagnostic laboratory. SIGNIFICANCE AND IMPACT OF THE STUDY: The performance of HSGME saves up to 90% time, material and energy costs, as well as laboratory hazardous wastes including carcinogenic agents used for visualizing DNA bands.

Analysis of the immune response to Yersinia enterocolitica serotype-O:9-released proteins by immunoblot and ELISA.

The immune response towards the released Yersinia enterocolitica outer membrane proteins (Yop) was analysed by immunoblotting and ELISA using a rabbit experimental model. Rabbits orogastrically or intravenously infected with the virulent (plasmid-bearing) Y. enterocolitica O:9 W836 strain developed a significant response by (IgG) antibodies to the released proteins having molecular weights of 51 (YopH) and 41 (LcrV) kDa, respectively. However, only in animals infected via the orogastric route were specific antibodies of the IgG class found against plasmid-encoded polypeptides of 35 (YopN) and 20 (YopQ) kDa. These results suggest that the expression of Yop in vivo may be conditioned by the route of infection used. Using ELISA, a significant response by IgG-class antibodies to YopH protein was evident in the sera from rabbits both orogastrically and intravenously infected with the virulent (pYV+) Y. enterocolitica O:9 W836 strain. By contrast, no specific antibodies to this antigen were detected in sera of rabbit infected with an avirulent (plasmid-cured) derivative (pYV-) strain. Accordingly, this protein could be very useful as an antigen in ELISA for serological diagnosis of infections caused by enteropathogenic strains of Yersinia spp.