Multiple Semi-nested Polymerase Chain Reaction Detection In Cerebrospinal Fluid Of Bacterial 16s Rrna Genes

Objective To design and establish a new method to dectect patho-genic bacterial 16S rRNA gene in cerebrospinal fluid (CSF) using mutiplex semi nested PCR. Method Acoording to the analysis of the conservative and variable regions in bacterial 16S rRNA genes , we designed universal primers for all bacteria and specific primers for most gram-positive and gram-negtive bacteria . All primers were added into the same reaction systems successively of a two-step PCR assay to amplify the differient bacterial 16S rRNA gene in 62 CSF samples, and compared with common culture method；The sesitivity and the specificity were detected at the same time. Results Both E. coli and S. aureus amplified a 1032bp DNA fragment; In addtion,specific fragments of 336bp and 127bp were amplified in S.aureus and E.coli respectively ；HPV,C. albicans ,huaman genomic DNA,and water had no specific amplicon. The detection limit for E.coli was 8CFU/ml. 62 CSF samples were decteced by both the multiplex semi-nested PCR and conventional bacteriologic method ,the comparision revealed sensitivity,specificity, positive and negative predictive values of 93.8%,95.7%,88.2% and 97.8% res-pctively for PCR. Conclusion The result suggests that the multiplex semi-nested PCR we established is a more sesitive,specific and rapid method for clinical laboratroy to detect bacterial pathogenes.