Amplification of bacterial DNA using highly conserved sequences: automated analysis and potential for molecular triage of sepsis.
OBJECTIVE
The clinical diagnosis of sepsis remains difficult, particularly in the young child, and would be improved by a rapid and reliable method for identification of bacteria in blood and other body fluids. Polymerase chain reaction (PCR) amplification of highly conserved DNA sequences found in all bacteria would permit fast and sensitive determination of the presence of bacteria in clinical specimens.


METHODS
A primer pair for highly conserved regions of bacterial DNA encoding 16S ribosomal RNA (rDNA) was utilized for PCR amplification. PCR products were analyzed by gel electrophoresis, and, after modification of the primers, by an automated 96-well plate reader.


RESULTS
rDNA was amplified from 12 different species of bacteria, including Gram-negative and -positive organisms. No signal was observed when total human DNA was used as template. Colorimetric analysis of amplified sequences using a 96-well format was also successful.


CONCLUSIONS
We conclude that a single primer pair designed to anneal to a highly conserved region of bacterial DNA can amplify DNA specimens from a variety of different bacteria, while not amplifying human DNA. Such a molecular genetics approach can be fully automated with existing robotic technology. Because of speed, sensitivity, and cost, molecular triage of patients with signs and symptoms of possible bacterial infection will decrease morbidity and mortality among those with unrecognized bacteremia who are managed as outpatients, and will dramatically reduce hospital expenses from individuals who are admitted and are not bacteremic.
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.
K M
KM
McCabe
G
G
Khan
Y H
YH
Zhang
E O
EO
Mason
E R
ER
McCabe
D001322:Autoanalysis; D002648:Child; D002675:Child, Preschool; D003124:Colorimetry; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis*; D004586:Electrophoresis; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D016133:Polymerase Chain Reaction / Q000379:methods*; D018805:Sepsis / Q000175:diagnosis*; D014218:Triage
D017931:DNA Primers; D004269:DNA, Bacterial