Diagnosis of Bacteremia by Universal Primer of Eubacteria

Uh, Young; Lee, Chang Hoon; Lee, Mi Kyung; Park, Il Gu; Park, Deok Woo; Yoon, Kap Jun

Abstract

BACKGROUND: The prompt detection of bacteremia continues to be one of the most important responsibilities of clinical microbiology. But clinical diagnosis of bacteremia remains difficult, particularly in the neonates and young children. And fastidious bacteria with specific growth requirements or bacteria requiring longer incubation period are apt to be negative results in blood cultures. Therefore, polymerase chain reaction (PCR) amplification which targets the highly conserved DNA sequences found in all eubacteria would permit fast and sensitive determination of the presence of bacteria in blood.
METHODS
A primer pair (DG74, RW01) for highly conserved regions of bacterial DNA encoding 16S ribosomal RNA (rRNA) was utilized for PCR amplification. PCR results were compaired with blood cultures and PCR products were digested with SmaI restriction enzyme for cutting of recognition site.
RESULTS
Among 44 blood specimens which organisms were isolated by blood culture, 41 samples were positive for PCR, and 3 samples which C. albicans, P. aeruginosa, and gram- positive bacillus isolated were negative. No signal was observed when blood obtained from person without clinical sign and or symptoms of bacteremia. All 41 PCR products (371 bp) were cutted in two DNA fragments (161 bp, 210 bp) by SmaI enzyme.
CONCLUSIONS
We concluded that a single primer pair designed to anneal to a highly conserved region of bacterial DNA can amplify DNA specimens from different bacteria, while not amplifying human DNA. Because of early detection, molecular trial of patients with signs and symptoms of possible bacterial infection will decrease morbidity and mortality with bacteremia, this approach may make it possible to identify new, nonculturable bacterial pathogens. Furthermore, if we use the specific primers for gram positive or negative bacteria in PCR method, it would be a more useful diagnostic tool for the clinicians.