PCR AMPLIFICATION AND CYCLE DNA-SEQUENCING ANALYSIS OF THE CHLAMYDIA-TRACHOMATIS ELONGATION-FACTOR TU GENE

Based on the elongation factor Tu (EF-Tu) gene of Chlamydia trachomati s, a pair of oligonucleotide primers CTUFU and CTUFD, were designed to amplify a specific target fragment of 931 bp. The PCR assay could det ect C. trachomatis in cervical smear specimens obtained from sex worke rs undergoing routine examination in an STD clinic. Distinct target ba nds were also amplified from at least 10 ng of positive control DNA sa mples from cultured cells infected with C. PCR with these primers coul d differentiate C. from eight non-chlamydial trachomatis. trachomatis bacterial species. Further verification could be obtained from the non -digestion of C. PCR products by trachomatis MspA1I restriction endonu clease, in contrast to the digestion of the non-specific PCR products of Klebsiella and Bacillus. Direct cycle DNA sequencing of similar to 450 bp of the PCR products of four C. trachomatis isolates revealed co mplete identity of one isolate with the known sequence of serovar F, w hile the other three isolates harboured three phenotypically silent po int mutations at codons 96, 305 and 312 of the EF-Tu gene. The sequenc e analyses confirm the authenticity of the target bands, reiterate the conservation and role of the EF-Tu gene in protein biosynthesis, and indicate the utility of the primers for the rapid detection of C. trac homatis.