[Establishment of quantifying and typing analysis of 16S rRNA gene by real-time PCR].
OBJECTIVE
To explore a new method of rapid and reliable diagnosis of bacterial infectious diseases such as purulent meningitis and septicemia.


METHODS
A pair of universal primers and a set of probes (including universal fluorescence probe, Gram-positive probe and Gram-negative probe) were designed based on the bacterial highly conserved region of 16S rRNA gene. By using the FQ-PCR method, 12 standard strains, 23 clinical cultural isolations and the controls such as HBV, Cryptococcus histolyticus, Blastomyces albicans and human DNA were detected with the three kinds of probes. The correlation among the results of the three kinds of probes detection was analyzed.


RESULTS
The determination of 16S rRNA gene with FQ-PCR was a highly specific and sensitive method and not cross-reactive with human DNA, virus or fungi. The least amount of 10 copies of 16S rRNA gene which corresponded to 2 bacteria could be detected with FQ-PCR. Twelve standard strains and 23 clinical cultural isolations were detected by FQ-PCR with the three kinds of probes mentioned above. All samples presented positive results using the universal probe. The results of 16S rRNA gene detected by the Gramjpositive probe were positive to the 18 G+ strains. The results of 16S rRNA gene detected by the Gram-probe were positive to the 17 G- strains.


CONCLUSIONS
The FQ-PCR technique was established for bacteria quantifying and typing using the universal primer and the double type probes. This method was convenient and rapid in detecting, quantifying and typing bacteria, with a high specificity and sensitivity.
Shu
Xiao-Li
XL
Department of Central Laboratory, Children's Hospital Affiliated to Medical College, Zhejiang University, Hangzhou 310003, China.
Wu
Yi-Dong
YD
Shang
Shi-Qiang
SQ
D005453:Fluorescence; D020459:Genes, rRNA; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*
D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S