Abstract
BackgroundSeveral 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.ResultsIn 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.ConclusionsThe 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.
Highlights
Several Multilocus Sequence Typing (MLST) schemes have been developed for Chlamydia trachomatis
Using stringent criteria during primer designing, we selected primers that gave significantly better scoring by Clone Manager 9.0 (Sci-Ed Software)
This study presents for the first time data about Israel genovars and sequence types (ST)’s distribution among C. trachomatis
Summary
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. Poor results have been obtained with Bom’s MLST scheme in clinical samples with low concentrations of Chlamydia DNA. Chlamydia trachomatis is a Gram-negative, obligate intracellular bacterium, responsible for a wide range of diseases [1]. Since the advent of sequencing, sequence analysis of the MOMP gene (ompA), which encoded by nearly 1200 bp, has been widely used for epidemiological studies [20]. The genome is characterized by a low level of genetic diversity among variants (< 2% of the genome), and the ompA genotype classification strongly correlates with tissue tropism and disease outcome [26, 27]
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