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Bom\u2019s MLST scheme for MLST is based on nested PCR amplification and sequencing of five hypervariable genes and <jats:italic>ompA<\/jats:italic>. In contrast to other Chlamydia MLST schemes, Bom\u2019s 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\u2019s MLST scheme in clinical samples with low concentrations of Chlamydia DNA.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>In this work, we present an improved version of the scheme that is based on the same genes and MLST database as Bom\u2019s 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 <jats:italic>in-silico<\/jats:italic> 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 &amp; 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 <jats:italic>improved MLST<\/jats:italic> version was used to obtain a genovars distribution in patients attending an STI clinic in Tel Aviv.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>The newly developed MLST version showed great improvement of assay results for samples with very low concentrations of <jats:italic>Chlamydia<\/jats:italic> DNA. A similar concept could be applicable to other MLST schemes.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s12866-020-02077-y","type":"journal-article","created":{"date-parts":[[2021,1,18]],"date-time":"2021-01-18T15:02:50Z","timestamp":1610982170000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["High-resolution multilocus sequence typing for Chlamydia trachomatis: improved results for clinical samples with low amounts of C. trachomatis DNA"],"prefix":"10.1186","volume":"21","author":[{"given":"Shlomo","family":"Pilo","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gal","family":"Zizelski Valenci","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mor","family":"Rubinstein","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lea","family":"Pichadze","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yael","family":"Scharf","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zeev","family":"Dveyrin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Efrat","family":"Rorman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6726-5052","authenticated-orcid":false,"given":"Israel","family":"Nissan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,1,18]]},"reference":[{"issue":"8","key":"2077_CR1","doi-asserted-by":"publisher","first-page":"464","DOI":"10.1016\/j.tim.2014.04.013","volume":"22","author":"NL Bachmann","year":"2014","unstructured":"Bachmann NL, Polkinghorne A, Timms P. 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