Quint Koen D, van Doorn Leen-Jan, Kleter Bernhard, de Koning Maurits N C, van den Munckhof Henk A M, Morre Servaas A, ter Harmsel Bram, Weiderpass Elisabete, Harbers Gonneke, Melchers Willem J G, Quint Wim G V
DDL Diagnostic Laboratory, Fonteijnenburghlaan 7, 2275 CX Voorburg, The Netherlands.
J Mol Diagn. 2007 Nov;9(5):631-8. doi: 10.2353/jmoldx.2007.070011. Epub 2007 Sep 14.
Chlamydia trachomatis (Ct) comprises distinct serogroups and serovars. The present study evaluates a novel Ct amplification, detection, and genotyping method (Ct-DT assay). The Ct-DT amplification step is a multiplex broad-spectrum PCR for the cryptic plasmid and the VD2-region of ompl. The Ct-DT detection step involves a DNA enzyme immunoassay (DEIA) using probes for serogroups (group B, C, and intermediate) and the cryptic plasmid, permitting sensitive detection of 19 Ct serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3) without any cross-reactivity with other Chlamydia species and pathogenic bacteria or commensal organisms of the genital tract. Ct-positive samples are analyzed by a nitrocellulose-based reverse hybridization assay (RHA) containing probes for the 19 different serovars and for the cryptic plasmid. The sensitivity of the PCR-DEIA on clinical specimen is equivalent to that of the Cobas TaqMan assay [kappa = 0.95 (95% confidence interval = 0.92 to 0.99)]. Using the RHA, 98% of the Ct-DT detection step-positive samples could be typed. Analysis of cervical swabs from Uganda and The Netherlands revealed that the most common serovars in Uganda are G/Ga (45%), E (21%), K (13%), and F (8%), and in The Netherlands serovars E (38%), F (23%), G/Ga (11%), and D/Da (7%) were most common. Thus, multiplex broad-spectrum PCR in combination with DEIA and RHA permits highly sensitive and specific detection and identification of Ct serovars.
沙眼衣原体（Ct）由不同的血清群和血清型组成。本研究评估了一种新型的Ct扩增、检测和基因分型方法（Ct-DT检测法）。Ct-DT扩增步骤是针对隐蔽质粒和外膜蛋白主要外膜蛋白环（omp）的可变区2（VD2）区域的多重广谱聚合酶链反应（PCR）。Ct-DT检测步骤涉及一种DNA酶免疫测定法（DEIA），该方法使用针对血清群（B群、C群和中间群）和隐蔽质粒的探针，能够灵敏地检测19种Ct血清型（A、B/Ba、C、D/Da、E、F、G/Ga、H、I/Ia、J、K、L1、L2/L2a和L3），且与其他衣原体物种、致病细菌或生殖道共生生物无任何交叉反应。Ct阳性样本通过基于硝酸纤维素的反向杂交分析（RHA）进行分析，该分析包含针对19种不同血清型和隐蔽质粒的探针。PCR-DEIA对临床标本的敏感性与Cobas TaqMan检测法相当[kappa = 0.95（95%置信区间 = 0.92至0.99）]。使用RHA，98%的Ct-DT检测步骤阳性样本能够进行分型。对来自乌干达和荷兰的宫颈拭子分析显示，乌干达最常见的血清型是G/Ga（45%）、E（21%）、K（13%）和F（8%），而在荷兰，血清型E（38%）、F（23%）、G/Ga（11%）和D/Da（7%）最为常见。因此，多重广谱PCR结合DEIA和RHA能够实现对Ct血清型的高度灵敏和特异的检测与鉴定。
