| Background Following the advent of aging society and a increasing number of people with HIV infection, people of low immunological competence are becoming more and more, which lead to many deaths, especially for neonatal death due to sepsis. According to report from National Nosocomial Infection Surverllance System, incidence of bacteremia is 48.56 per billion, which accounts for 1.3% in nosocomial infection. In fact, severe sepsis and septic shock is the 10th leading cause of death in the United States. To lower mortality, rapid detection of pathogens and antibacterial therapy are required. However, conventional culture method, as a gold standard, is time-consuming, laborious and not fit for fastidious pathogens, such as S. pneumonia. Besides, some pathogens can't grow due to the effect of antibiotics. Therefore, it is desirable to explore a simple and sensitive method to detect pathogenic bacteria in clinical blood samples. rRNA sequence analysis has been used to clarify the taxonomic affinities of a wide range of taxa and as a powerful tool for the detection of pathogenic bacteria.Capillary electrophoresis which was developed on the basis of slab gel electrophoresis was used to separate biomacromolecule in 1980s. Meanwhile, single strand conformation polymorphism was developed as a genotyping method by Orita et al. and Kuypers et al. combined CE with SSCP to detect gene mutation. This technology (CE-SSCP) has been widely used for mutation detection, bacteria identification, analysis of antimicrobial activities, SNP genotyping and mRNA quantification due to its advantages such as simplicity, sensitivity, and speed. In this study, we detected the common pathogens in blood culture, including S.aures, E.coli, P.aeruginosa, S.epidermidis, K.pneumonia, E.faecalis and S.pneumonia using CE-SSCP combined with PCR to establish a novel method for identification of pathogens.Method 1. The common pathogens of blood infection were chosen, whose genomic DNA were obtained by entering NCBI. Multi-sequences aligment were done for searching conserved regions, where universe primers were designed. 2. Genomic DNA isolated from each standard strain were used for amplification. The resulting peak products were analyzed by CE-SSCP to establish electrophoretogram. Sensitivity of the method was also studied. 3. Genomic DNA isolated from clinic strains were anylyzed by CE-SSCP whose electrophoretogram were compared with those from standard strains. Meanwhile, CE-SSCP was also compared with conventional culture.Result 1. The universal primers from conserved regions of 16S rDNA and 23S rDNA were successfully amplified target fragments with the expected size without obvious non-specific amplification or dimers. 2. When PCR products of 16S rDNA were analyzed by CE-SSCP, there were different data point (Cv≥1.33%) and corresponding fragment length (Cv≤0.046%) corresponding to seven kinds of pathogens, which were S. aures (317.40-317.61), E. coli (309.83-309.99), P. aeruginosa (305.30-305.59), S. epidermidis (316.34-316.63), K. pneumoniae (307.76-307.96), E. faecalis (327.28-327.57) and S. pneumonia (315.86-315.62), respectively. 16S-23S rDNA also had different electophoretograms. When they were encoded by machine code, it became more convenient to distinguish all of the pothogens. 3. After detection of clinic strains, the fragements were in accordance with those from standard strains. Compared with conventional microbiological method, the coincidence rate was 94.8% and its sensitivity was 18 CFU/ml.Coclusion 1. Universal primers could successfully amplify seven kinds of pathogens without obvious non-specific amplification or dimers. 2. When products of 16S rDNA were analyzed by CE-SSCP, seven kinds of pathogens could be differentiated simultaneously, which was of significance to dignosis of polymicrobial infection. According to the studies of different criterion, corresponding fragment length was superior to data point. Machine code was led to encode electrophoretogram, which made bacteria detection more convenient and provided some mentality of designing automatic sofeware. 3. This test method allowed simultaneous identification of seven kinds of pathogens fast, sensitively and conveniently within 4-6h, which laid a good foundation for its clinical application. |
