PCR amplification and cycle DNA sequencing analysis of the Chlamydia trachomatis elongation factor Tu gene

Abstract

Based on the elongation factor Tu (EF-Tu) gene of Chlamydia trachomatis, a pair of oligonucleotide primers CTUFU and CTUFD, were designed to amplify a specific target fragment of 931bp. The PCR assay could detect C. trachomatis in cervical smear specimens obtained from sex workers undergoing routine examination in an STD clinic. Distinct target bands were also amplified from at least 10ng of positive control DNA samples from cultured cells infected with C. trachomatis. PCR with these primers could differentiate C. trachomatis from eight non-chlamydial bacterial species. Further verification could be obtained from the non-digestion of C. trachomatis PCR products by MspA1I restriction endonuclease, in contrast to the digestion of the non-specific PCR products of Klebsiella and Bacillus. Direct cycle DNA sequencing of ∼450bp of the PCR products of four C. trachomatis isolates revealed complete identity of one isolate with the known sequence of serovar F, while the other three isolates harboured three phenotypically silent point mutations at codons 96, 305 and 312 of the EF-Tu gene. The sequence 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. trachomatis.