A molecular biological study on identification of common septicemia bacteria using 16s–23s rRNA gene spacer regions

Abstract

In the search for a rapid and reliable method for identification of bacteria in blood and cerebrospinal fluid, we developed a unified set of primers and used them under polymerase chain reaction (PCR) to amplify the spacer regions between the 16s and 23s genes in the prokaryotic rRNA genetic loci. Spacer regions within these loci showed a significant level of length and sequence polymorphism across most of the species lines. A generic pair of priming sequences was selected from highly conserved sequences in the 16s and 23s genes occurring adjacent to these polymorphic regions. This single set of primers and reaction conditions were used for the amplification of the 16s–23s spacer regions for 61 strains of standard bacteria and corresponding clinical isolates belonging to 20 genera and 27 species, including Listeria, Staphylococcus and Salmonella species, et al. When the spacer amplification products were resolved by electrophoresis, the resulting patterns could be used to distinguish most of the bacteria species within the test group, and the amplification products of the clinical isolates clustered at the standard species level. Some species presenting similar pattern were further analyzed by HinfI or AluI digestion or DNA clone and sequences analysis in order to establish the specific 16s–23s rRNA gene spacer regions map. Analysis of 42 blood specimens from septicemic neonates and 6 CSF specimens from suspected purulent meningitis patients by bacterial culture and PCR-RFLP (Restriction Fregament Length Polymorphism) showed that 15 specimens of blood culture were positive (35.7%) in the 42 septicemic neonates; 27 specimens were positive(64.2%) by PCR, and that the positive rate by PCR was significantly higher than that by blood culture(P < 0.01). Among the 6 CSF specimens, one specimen found positive by blood culture was also positive by PCR, two found negative by blood culture showed positive by PCR; all three were S. epidermidis according to the DNA map. One C. neoformans found positive by blood culture showed negative by PCR. The remaining two specimens were both negative by PCR and blood culture. These results indicated that the method of detecting bacterial 16s–23s rRNA spacer regions using PCR and RFLP techniques was rapid, sensitive and specific in the detection of bacterial infections; and so, has very important application in the clinical diagnosis of sepsis in neonates.