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, particu larly in the young child, and would be improved by a rapid and reliabl e method for identification of bacteria in blood and other body fluids . Polymerase chain reaction (PCR) amplification of highly conserved DN A sequences found in all bacteria would permit fast and sensitive dete rmination of the presence of bacteria in clinical specimens. Methodolo gy. A primer pair for highly conserved regions of bacterial DNA encodi ng 16S ribosomal RNA (rDNA) was utilized for PCR amplification. PCR pr oducts were analyzed by gel electrophoresis, and, after modification o f the primers, by an automated 96-well plate reader. Results. rDNA was amplified from 12 different species of bacteria, including Gram-negat ive and -positive organisms. No signal was observed when total human D NA was used as template. Colorimetric analysis of amplified sequences using a 96-well format was also successful. Conclusions. We conclude t hat a single primer pair designed to anneal to a highly conserved regi on of bacterial DNA can amplify DNA specimens from a variety of differ ent bacteria, while not amplifying human DNA. Such a molecular genetic s approach can be fully automated with existing robotic technology. Be cause of speed, sensitivity, and cost, molecular triage of patients wi th signs and symptoms of possible bacterial infection will decrease mo rbidity and mortality among those with unrecognized bacteremia who are managed as outpatients, and will dramatically reduce hospital expense s from individuals who are admitted and are not bacteremic.