[Molecular diagnosis of the specific DNA patterns of 16S-23S rRNA gene of bacteria].

OBJECTIVE: To establish the specific 16S-23S rRNA gene spacer regions pattern in different bacteria using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequences analysis. METHODS: A pair of primers were selected from highly conserved sequences adjacent to the 16S-23S rRNA spacer region. Bacterial DNA of sixty-one strains of standard bacteria and corresponding clinical isolates representative of 20 genera and 27 species was amplified by PCR, and further studied by RFLP, DNA cloning and sequences analysis. Meanwhile, all specimens were examined by bacterial culturing and PCR-RFLP analysis. RESULTS: The 27 different standard strains showed one, two, three or more than three bands. The sensitivity of PCR reached 2.5 colony-forming unit (CFU), and there was no cross reaction to the human, fungal or viral genomic DNAs. Fifteen species could be distinguished immediately by PCR, while another 10 species were further identified by Hinf I or Alu I digestion. Klebsiella pneumoniae (Kp) and Enterococcus durans (Ed) could not be differentiated from each other by Alu I or Hinf I digestion. The spacer sequences of the Kp and Ed were 908 bp and 909 bp, respectively, and they differed only at the site of the 779th nucleotide. The former was G, and the latter was A. The 760 - 790 bp sequence of Kp was as follows: CGACTGCACCGCCTCCTAC / GGCCGCGTATTC. The 760 - 790 bp sequence of Ed was as follows: CGACTGCAC CGCCTCCTAC / AGCCGCGTATTC. Only one enzyme XmaIII, could discriminate the two. The cleaving site of XmaIII is C downward arrow GGCCG. Kp DNA was cleaved into 778 bp and 130 bp fragments, while E. durans was not. Of 42 specimens with suspected septicemia, 15 were positive (35.7%) on blood culture, and 27 on PCR (64.29%). The positive rate of PCR was significantly higher than that of blood culture (P < 0.01). Of the six CSF specimens, one was positive for Staphylococcus epidermidis (Se) on culture as well as by PCR, while two specimens which were negative on cultures were positive by PCR and were diagnosed as Se according to its DNA pattern. One specimen was culture-positive for Cryptococcus neoformans (Cn) but was negative by PCR. The other two specimens were negative by both PCR and culture. Fifteen blood samples from healthy children were negative by both blood culture and PCR. CONCLUSIONS: The method of detecting bacterial 16S-23S rRNA spacer regions using PCR-RFLP techniques was specific, sensitive, rapid and accurate in detecting pathogens in clinical bacterial infections.

[Study on the bacterial identification using 16S-23S rRNA gene spacer regions]

OBJECTIVE: To examine the use of PCR utilizing 16S-23S rRNA gene spacer regions in the identification of bacteria. METHODS Primers used in PCR were designed by using the target sequences from the genes encoding 16S-23S rRNA spacer regions. PCR was used for the detection of different standard and clinical bacterial strains. RESULTS Characteristic DNA maps were present after using the PCR to identify 27 standard strains from 27 species. The maps could be directly used for classification of the tested bacterial strains or further differentiated by RFLP. The sensitivity of the PCR may be as high as 2.5 CFU/ml. No non-specific amplification products were observed when using DNA from human PBMC funguses or viruses as templates. Thirty-two strains of bacteria isolated from clinical strains showed DNA maps similar to the DNA maps amplified from standard strains. CONCLUSION The PCR detection of bacteria using 16S-23S rRNA gene spacer regions is sensitive, rapid, specific and accurate for identification of bacteria and provides a new rapid method for determining the clinical diagnosis and the etiology of sepsis.