Storm Martin, Gustafsson Ingegerd, Herrmann Björn, Engstrand Lars
Uppsala University, Department of Medical Sciences, Clinical Bacteriology, Uppsala, Sweden.
J Microbiol Methods. 2005 Jun;61(3):361-7. doi: 10.1016/j.mimet.2004.12.015. Epub 2005 Jan 20.
Pharmacodynamic knowledge about Chlamydia trachomatis exposed to antibiotics is hampered due to methodological limitations. We have developed a quantitative real-time PCR method (qRT-PCR) for determination of viable C. trachomatis. The method measures specific RNA transcripts of omp2 (omcB) as an expression of viable C. trachomatis. Two clinical isolates (one strain derived from a patient with recurrent symptoms despite doxycycline treatment) were cultured in McCoy cells and exposed to doxycycline at concentrations of 0.0078-64 mg/L. MIC values were evaluated microscopically by immunofluorescence (IF) and by qRT-PCR performed on cDNA prepared from the total RNA. The MIC for two C. trachomatis strains were determined to 0.016 and 0.031 mg/L by both qRT-PCR and IF. The qRT-PCR assay enabled MIC determinations without subjective evaluation, which is a problem when visually evaluating inclusions. The presented qRT-PCR is a suitable method for MIC determination of C. trachomatis. It has the advantage of giving quantitative measurements of chlamydial RNA levels and the method is useful in pharmacodynamic studies of C. trachomatis.
由于方法学上的局限性，关于暴露于抗生素下的沙眼衣原体的药效学知识受到了阻碍。我们开发了一种定量实时PCR方法（qRT-PCR）用于测定活的沙眼衣原体。该方法测量omp2（omcB）的特定RNA转录本作为活的沙眼衣原体的一种表达。将两株临床分离株（其中一株来自尽管接受强力霉素治疗仍有复发症状的患者）在 McCoy 细胞中培养，并暴露于浓度为 0.0078 - 64 mg/L 的强力霉素中。通过免疫荧光（IF）显微镜评估以及对从总RNA制备的cDNA进行qRT-PCR来评估MIC值。通过qRT-PCR和IF测定的两株沙眼衣原体菌株的MIC均为0.016和0.031 mg/L。qRT-PCR测定法无需主观评估即可确定MIC，而在目视评估包涵体时这是一个问题。所呈现的qRT-PCR是一种适用于测定沙眼衣原体MIC的方法。它具有对衣原体RNA水平进行定量测量的优势，并且该方法在沙眼衣原体的药效学研究中很有用。
