| Gram-positive cocci are the common pathogens causing human infectious diseases. In the last 10 years, the incidence of gram-positive cocci infections is increasing. Currently, the 3 most common causes of nosocomial bloodstream infections (BSIs) are coagulase-negative staphylococci, Staphylococcus aureus, and enteroccci. Many gram-positive cocci associated with nosocomial infections are now resistant to commonly used antibacterial agents. Methicillin-resistant staphylococci (MRS), vancomycin- resistant enterococci (VRE), and penicillin nonsusceptible Streptococcus pneumoniae (PNSSP) is of particular concern. Infections caused by resistant pathogens are associated with significantly higher mortality rate than susceptible pathogens, and options for effective antimicrobial therapy are becoming increasingly limited. Furthermore, the absence of effective therapy will cause mortality to increase. Accurate, early etiological diagnosis of bacterial infection leads to appropriate patient management, and allowing the rational use of antibiotics. This in turn reduces side effects for the patient, decreases the cost and may prevent the spread of drug-resistance. Conventional diagnostic method contain the culture of bacteria from clinical specimens and their subsequent antimicrobial susceptibility testing, thus, it need more time to provide a result (3~5day). With the advance in molecular biology and bacterial genomics, molecular methods have become established as accepted methods for the detection of causal agents of infection. Base on the analysis of 23S rRNA gene sequences and other genes sequence association with antimicrobial resistance (mecA, vanA, vanB, and pbp2b), we established a nucleic acid-based detection systems now offer rapid and sensitive methods to detect the presence of resistant microorganisms. It could provide identification and susceptibility data of pathogens in 6 hours, and may be helpful for the early etiological diagnosis and treatment of severe infections.Part 1 Study on molecular method to identify common pathogensThe rRNA genes are essential for the survival of all organisms and are highly conserved in bacteria. There are three genes, which make up the rRNA functionality: the 5S, 16S, and 23S rRNA genes. Highly variable portions of the rRNA sequence contain signatures unique for each bacterium, as well as useful information about therelationships between different bacteria. Furthermore, certain conserved regions of sequence are also found in all known bacteria. Broad-range PCR primers may then be designed to recognize these conserved bacterial rRNA gene sequence and used to amplify variable or diagnostic regions. After sequencing or hybridization, bacteria may be identified. The 16S rRNA gene has historically been most commonly employed; However, more recently, employment of 23 S rRNA gene has become popular. To analysis the sequence difference among the 23 S rRNA genes from common bacteria. 23 S rRNA genes from 13 common bacteria were amplified with a broad-range PCR primer, and products were sequencing, data were analysised with DNAStar soft. To explore the possible use 23 S rRNA gene in detecting or identifying bacteria. First, a primer, which could recognize the gram-positive cocci conserved region, was designed. Combination with broad-range PCR primers, a novel PCR- gel electrophoresis (PCR-GE) method was established. After amplifying and gel electrophoresis, gram-negative bacteria presented one band and gram-positive presented two bands. So the method could not only detect bacteria, but also discriminate gram-positive cocci and gram-negative bacteria; Second, after oligonucleotide probes being designed and broad-range primers being labeled with Dig, we established a PCR-reverse hybridization assay, a test that permits identification of common bacteria by means of species-specific probes. The efficacy of PCR-GE and PCR-reverse hybridization assay were evaluated with clinical isolates, and appear to be a sensitive, specific, and repeatability tool for detection or identify common bacteria. A... |
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