The Application Of Quantifying And Typing With The 16S RRNA Genes Real-Time PCR In The Surgical Bacterial Infectious Diseases

Objective To explore a rapid and reliable method of bacteriological diagnosis in the diseases of surgical bacterial infection.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 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,and analyzing the correlation among the results of the three kinds of probes detection.By using FQ-PCR method,102 specimens of ascites or pus collected from the patients of acute appendicitis,acute peritonitis,acute indigitation,and soft tissue abscess etc.were detected with the Gram-positive probe and Gram-negative probe.These specimens were sent to laboratory for bacteria culture at the same time.Results The 16S rRNA gene FQ-PCR was a highly specific and sensitive method and not cross-reactive with human DNA,virus or fungi.At least,10 copies of 16S rRNA gene which is corresponding to 2 bacteria could be detected with FQ-PCR.12 standard strains and 23 clinical cultural isolations were detected by FQ-PCR with all three kinds of probes.All samples presented positive results by using universal probe,G~+ Probe was positive to the 18 G~+ strains,G~- Probe was positive to the 17 G~- strains,and vice versa.The coincident rate was 100%.In the detectings of infectious ascites specimens,the FQ-PCR was positive at the rate 82.5%,the bacteria culture was positive at the rate of 32.5%.In the detectings of pus specimens detectings,FQ-PCR was positive at the rate of 100%,the bacteria culture was positive at the rate of 50%. Conclusions Establishing the FQ-PCR technique for bacteria quantifying and typing by using the universal primer and the double typied probes.This method was convenient and rapid in detecting, quantifying and typing bacteria with high specificity and sensitivity.It has considerable value of rapid diagnosis.