Most C. trachomatis genotyping assays to-date are relatively complex despite targeting a low number of highly variable loci, such as the ompA gene. Additionally, the recent availability of whole genome sequences has shown that these regions may not correlate with the whole genome phylogeny and therefore may obscure epidemiological patterns.
We developed an efficient and low cost genotyping assay for C. trachomatis, based on genome-wide orthologous single nucleotide polymorphisms (SNPs), which divides the species into the major evolutionary lineages, and also discriminates the ompA variants.
Orthologous SNPs were defined by alignment of 65 C. trachomatis genome sequences representative of the known global diversity. SNP sets optimised for combinatorial resolving power were derived using the software Minimum SNPs. PCR amplifiable fragments containing the SNPs were developed. High resolution melting analysis (HRMA) procedures were used to discriminate variants of amplified fragments. The resolving power of HRMA was predicted in silico, and then determined in practice.
In silico analyses predicted that HRMA of just two amplified fragments, nucleated by two SNPs, in Jali1891 and ompA, would resolve C. trachomatis into 15 groups. Further, the groups were predicted to be highly concordant with the major lineages in orthologous SNPs-based phylogeny, and also with ompA serovars. HRMA assays were developed, and tested using 35 cultured isolates of known genome sequence, representing much of C. trachomatis diversity. The results were in accordance with in silico predictions. Preliminary experiments indicate that the method may be used for direct analysis of clinical specimens.
A method for resolving C. trachomatis into 15 groups highly concordant with the major phylogenetic lineages and ompA serotypes has been developed. The method requires two PCR reactions followed by HRMA, using a real time PCR platform. The HRMA format allows rapid and high-throughput analysis suitable for large epidemiological studies.