Rapid Detection And Identification Of Pathogen In Body Fluid Using Universal Primers And RELP

Objective To establish a method to detect and identify pathogen in body fluids rapidly using universal primers PCR and restriction enzyme analysis.Method According to the analyses of the conservative and variable regions in bacterial 16S rRNA genes,we designed universal primers for all bacteria capable of amplifying DNA of various bacteria to determine whether there are bacteria.The PCR products were then digested with different restriction enzymes to finally identify clinical pathogen .Results All the bacteria amplified the same products of 1032bp after UP-PCR, but most of the restriction patterns of PCR products were different when digested by HaeJII. Products from S. aureus, S. epidermidis could not be digested byHaelll, while yielded different patterns when digested by Mnll. Products from S.pneumoniae, E. faetium yielded the same HaeJII digestion pattern, but be different when digesed by Alul . Products of E. coli, K. pneumoniae, S. marcescens, and E. cloacae also had the same Haelll digestion pattern, but different when digested with Ddel. The universal PCR could detect as few as 10CFU/ml E.coli or 250 CFU/ml S. aureus. Compared with culture method, the sensitivity of the universal PCR for detection and identification of bacteria from 102 body fluids was 92.3%.Conclution The results suggest that universal PCR coupled withrestriction enzyme analysis can detect and identify bacterial pathogensin clinical specimens.