A molecular biological study on identification of common bacteria causing septicemia by the analysis of 16S-23S rRNA gene spacer regions / 中华传染病杂志

ABSTRACT

Objective To establish the specific 16S 23S rRNA gene spacer regions map of different bacteria by PCR, RFLP(restriction fragment length polymorphism ),DNA clone and sequences analysis. Methods A pair of primer was selected from highly conserved sequences adjacent to the 16S 23S rRNA spacer region. The farget rRNA regions from 61 strains of standard bacteria and corresponding clinical isolates representing for 20 genera and 26 species were amplified by PCR,and thereafter analyzed RFLP, DNA clone and sequences analysis.Meanwhile, all the specimens were examined by bacterial culture and PCR RFLP analysis. Results 26 different standard strains presented one band,two bands,three bands and more than three bands respectively, the sensitivity of which reached 2.5 CFU and had no cross reaction to the human genomic DNA,fungus and virus.14 species could be distinguished immediately by PCR, other 10 species must be identified by further Hinf I or Alu I digestion. K.pneumoniae and E.durans differentiate only at the site of 779 th nucleotide according to the sequence analysis, and only one enzyme Xma III could discriminate them.15 specimens from 42 septicemic neonates were blood culture positive and the positive rate was 35.7%. However, 27 specimens were positive by PCR and the positive rate was 64.2%,which was significantly higher than that of the blood culture( P