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 organ failure syndrome, but there are currently no tests capable of detecting and quantitating translocation sequentially in humans. The purpose of this study was to develop a sensitive polymerase chain reaction (PCR) test to detect Escherichia coli (E. coli) DNA in the blood of animals after inducing bacterial translocation from the gut. DNA was extracted from blood and primers were used to amplify an 800-bp gene fragment of E. coli by 30-cycle PCR. Detection by southern blotting achieved a sensitivity of 10-100 organisms per 0.3 cc blood. Experimental groups included mice gavaged with 10(10) E. coli followed by 20% body surface area thermal injury, or no injury. Controls included burn only and no treatment groups. Blood was obtained by cardiac puncture 1 hr after burn. Cultures were done on blood samples from all groups. More animals in the burn/gavage group had positive bacterial cultures. All controls were culture negative. E. coli detection by PCR was 100% sensitive in culture positive animals with detection in the gavage/burn group higher than that in all other groups. PCR was negative for all mice without treatment. Several culture negative animals had detectable bacterial DNA by PCR. This highly sensitive and specific method can be used repeatedly to test the blood of patients for the presence of microbial DNA, which could be originating from the gut.
Kane
T D
TD
Department of Surgery, University of Cincinnati, Ohio 45267, USA.
Johnson
S R
SR
Alexander
J W
JW
Babcock
G F
GF
Ogle
C K
CK
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D017931:DNA Primers; D004269:DNA, Bacterial / Q000097:blood*; D004926:Escherichia coli / Q000302:isolation & purification* / Q000502:physiology; D004927:Escherichia coli Infections / Q000175:diagnosis* / Q000635:transmission; D005260:Female; D006801:Humans; D007422:Intestines / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial