Holt Deborah C, Andersson Patiyan, Buckley Cameron, Whiley David M, Giffard Philip M
Menzies School of Health Research, Charles Darwin University, Darwin, NT, Australia.
College of Health and Human Sciences, Charles Darwin University, Darwin, NT, Australia.
Methods Mol Biol. 2019;2042:87-122. doi: 10.1007/978-1-4939-9694-0_8.
CtGEM typing was developed to subdivide the bacterial species Chlamydia trachomatis on the basis of genome phylogeny and anatomical tropism. The rationale was facilitation of surveillance for ocular strains, although the method is applicable to essentially any C. trachomatis surveillance application that does not require high resolution. CtGEM is a double-locus genotyping method. The loci included in the assay were identified by computerized analysis of 65 complete genomes for resolution optimized sets of single nucleotide polymorphisms (SNPs). From this, two PCR amplifiable fragments were defined. One, rg1, is within a hypothetical gene annotated as Jali-1891 within the C. trachomatis B_Jali20 genome. The other, ofr, is within the ompA gene which encodes the major outer membrane protein. Variation in rg1 is conferred by two SNPs defining four haplotypes that exhibit concordance with genome phylogeny. Variation within ofr is more complex and allows for inference of ompA genotype, either to the level of single genotype, or group of closely related genotypes. Two CtGEM formats were developed. One is based on interrogation of the two loci by high resolution melting analysis (HRMA), and the other based on analysis of the loci by Sanger sequencing. The genotypes defined identify known ocular genotypes, discriminate known ocular genotypes from each other, discriminate the major phylogenetic lineages of the species, and discriminate all ompA genotypes with the exception of closely related variants within the genotypes H, I, J cluster. The Sanger sequencing format provides slightly more resolution that the HRMA format with respect to ompA genotype. An unusual aspect of this method is that all possible combinations of rg1 haplotype, and inferred ompA genotype(s) have been given CtGEM typing numbers. This includes types that at this time have not been shown to exist.
CtGEM分型方法是基于基因组系统发育和解剖嗜性对沙眼衣原体细菌物种进行细分而开发的。其基本原理是便于对眼部菌株进行监测，尽管该方法基本上适用于任何不需要高分辨率的沙眼衣原体监测应用。CtGEM是一种双位点基因分型方法。该检测中包含的位点是通过对65个完整基因组进行计算机分析来确定的，以优化单核苷酸多态性（SNP）的分辨率集。由此，定义了两个可通过PCR扩增的片段。一个是rg1，位于沙眼衣原体B_Jali20基因组中注释为Jali-1891的假设基因内。另一个是ofr，位于编码主要外膜蛋白的ompA基因内。rg1的变异由两个SNP赋予，这两个SNP定义了四种单倍型，这些单倍型与基因组系统发育一致。ofr内的变异更为复杂，可用于推断ompA基因型，无论是单个基因型水平，还是密切相关基因型组水平。开发了两种CtGEM格式。一种基于通过高分辨率熔解分析（HRMA）对两个位点进行检测，另一种基于通过桑格测序对位点进行分析。所定义的基因型可识别已知的眼部基因型，区分已知的眼部基因型，区分该物种的主要系统发育谱系，并区分除基因型H、I、J簇内密切相关变体之外的所有ompA基因型。就ompA基因型而言，桑格测序格式比HRMA格式提供的分辨率略高。该方法的一个不同寻常之处在于，rg1单倍型和推断的ompA基因型的所有可能组合都被赋予了CtGEM分型编号。这包括目前尚未被证明存在的类型。
