High-resolution multilocus sequence typing for Chlamydia trachomatis: improved results for clinical samples with low amounts of C. trachomatis DNA

Abstract

Background Several Multilocus Sequence Typing (MLST) schemes have been developed for Chlamydia trachomatis. Bom’s MLST scheme for MLST is based on nested PCR amplification and sequencing of five hypervariable genes and ompA. In contrast to other Chlamydia MLST schemes, Bom’s MLST scheme gives higher resolution and phylogenetic trees that are comparable to those from whole genome sequencing. However, poor results have been obtained with Bom’s MLST scheme in clinical samples with low concentrations of Chlamydia DNA. Results In this work, we present an improved version of the scheme that is based on the same genes and MLST database as Bom’s MLST scheme, but with newly designed primers for nested-1 and nested-2 steps under stringent conditions. Furthermore, we introduce a third primer set for the sequencing step, which considerably improves the performance of the assay. The improved primers were tested in-silico using a dataset of 141 Whole Genome Sequences (WGS) and in a comparative analysis of 32 clinical samples. Based on cycle threshold and melting curve analysis values obtained during Real-Time PCR of nested-1 & 2 steps, we developed a simple scoring scheme and flow chart that allow identification of reaction inhibitors as well as to predict with high accuracy amplification success. The improved MLST version was used to obtain a genovars distribution in patients attending an STI clinic in Tel Aviv. Conclusions The newly developed MLST version showed great improvement of assay results for samples with very low concentrations of Chlamydia DNA. A similar concept could be applicable to other MLST schemes.

Triple site sexually transmitted infection testing as a crucial component of surveillance for men who have sex with men: A prospective cohort study

“…Multi-locus sequence typing (MLST) of five CT hypervariable genes and the outer membrane protein A ( ompA ) genotyping were performed. 16 Molecular serotyping was determined by blast analysis of the ompA sequence.…”

Mpox proctitis as a likely predisposing factor for chlamydial perihepatitis in a male patient

“…Lymphogranuloma venereum (LGV) was identified by the Allplex Genital Ulcer Assay. The sample was further analysed by in-house Multi Locus Sequence Typing 3. The strain was identified as LGV ST23.…”

“…The sample was further analysed by in-house Multi Locus Sequence Typing. 3 The strain was identified as LGV ST23.…”

Unravelling Chlamydia trachomatis diversity in Amhara, Ethiopia: MLVA-ompA sequencing as a molecular typing tool for trachoma

“…Classic MLST is focused on sequence variation in loci that are not considered to be under selection pressure and are stable housekeeping genes [18,19] and is best suited to long-term and global epidemiology. Other MLST studies have used highly variable loci instead, to improve variant resolution on a smaller epidemiological scale [20][21][22]. MLVA provides a further alternative and uses variable number tandem repeats (VNTR), which have a higher rate of mutations [23] and can therefore provide a useful identifier of novel strains for small scale, local epidemiological studies [22].…”