DETECTION OF INTESTINAL BACTERIAL TRANSLOCATION USING PCR

Microbial translocation has been suspected to be a major contributing factor in the development of sepsis of unknown origin and multiple org an failure syndrome, but there are currently no tests capable of detec ting and quantitating translocation sequentially in humans, The purpos e of this study was to develop a sensitive polymerase chain reaction ( PCR) test to detect Escherichia coli (E. coli) DNA in the blood of ani mals after inducing bacterial translocation from the gut. DNA was extr acted from blood and primers were used to amplify an 800-bp gene fragm ent of E. coli by 30-cycle PCR, Detection by southern blotting achieve d a sensitivity of 10-100 organisms per 0.3 cc blood. Experimental gro ups included mice gavaged with 10(10) E. coli followed by 20% body sur face area thermal injury, or no injury, Controls included burn only an d no treatment groups. Blood was obtained by cardiac puncture 1 hr aft er burn. Cultures were done on blood samples from all groups, More ani mals in the burn/gavage group had positive bacterial cultures. All con trols were culture negative. E. coli detection by PCR was 100% sensiti ve in culture positive animals with detection in the gavage/burn group higher than that in all other groups. PCR was negative for all mice w ithout treatment. Several culture negative animals had detectable bact erial DNA by PCR, This highly sensitive and specific method can be use d repeatedly to test the blood of patients for the presence of microbi al DNA, which could be originating from the gut. (C) 1996 Academic Pre ss, Inc.