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.

Keywords: Chlamydia trachomatis; Genovar; MLST; Melting curve analysis; Multilocus; Sequence; Typing.

Fig. 1

Flowchart of the strategy that we utilized in our MLST primer designing

Fig. 2

Representative results for typical nested 2 step amplification and melting curve analysis for each of the hctB (CT046), CT058, CT144, CT172 and pbpB (CT682) genes, the left figure shows the melting curve and the right the amplification plot (multicomponent plot). A) typical successful nested 2 step PCR amplification of the ATCC strains: ATCC VR-885, ATCC VR-886, ATCC VR-878 and ATCC VR-901B. B) Typical successful and unsuccessful nested 2 step PCR amplification of clinical samples: G (Good) successful amplification, B (Bad) Unsuccessful amplification and M (Moderate). Unsuccessful amplification of CT058 of sample 298 versus positive control (a), Successful amplification of CT046 of sample 293 versus positive control (b), Unsuccessful amplification of CT172 of sample 230 versus positive control (c), and Moderate amplification of CT682 of sample 362 versus positive control (d)

Fig. 3

The correlation between DNA concentration (a) and threshold cycle (b, c) in the sample to sequencing successes are shown in the boxplots. c Correlation between threshold cycle to sequencing successes of ompA. Odds of success are high when CT≤33. Above this value, success rates decrease

Fig. 4

Genovars and MLST distribution of the 81 clinical samples from patients attending community clinic for STI’s and HIV in Tel-Aviv. a Serovar of 67 out of 81 urine samples (82%). b Integration of the genovars and MLST distribution of 52 out of 81 samples (64.2%). NIUD – Not In Uppsala Database, represent novel ST’s. ND- Not Determined. ST\ST - there are two options for sequence type (434\510, 56\370)