Preliminary Study On Oligo-Chips For Detecting Pathogens In Cerebrospinal Fluid

Objective To develop a preliminary oligo-chips method for detection pathogens in cerebrospinal fluid (CSF), and explore perfect protocol for the farther study.Methods According to the analysis of the conservative and variable regions in bacterial 16S rRNA genes, we designed a pair of universal primers that can amplify rDNA of all bacteria. After UP-PCR, we can find out whether there is bacterial infection in the sample. 10 kinds of specific Probe were designed by Primer Premier software. Probes for identification were tailed and then spotted onto a nylon membrane. DNA are isolated from each pathogen, and subjected to UP- PCR to amplify target fragments, which are labeled with bio-16-dUTP at the same time. All those fragments were denatured, and then hybridized to the all kinds of probes on nylon membrane and visualized by AKP labeled avidin. The sensitivity and specificity of the detection system was detected. A total of 32 CSF samples, which were verified the bacterial infection by the routine method, were tested by this methods.Results All bacteria we tested amplified a 372bp DNA fragment, it is sensitive to 10 CFU/mL when detecting E.coli. Every kind of pathogens only react to its corresponding probes fixed on nylon membranes, which show high specificity. The result of identifying 32 CSF clinical specimens show to that of routine method.Conclusion The method can screen out partial pathogens in CSF successfully, it is sensitive, rapid and specific. In addition, it could determine multifold pathogens through only one experiment. Therefore it is well worth of promoting in clinics, and lay the foundation for our farther research.