Jalal Hamid, Stephen Hannah, Curran Martin D, Burton Janet, Bradley Michelle, Carne Christopher
Clinical Microbiology & Public Health Laboratory, Addenbrooke's Hospital, Cambridge, UK.
J Clin Microbiol. 2006 Jan;44(1):206-13. doi: 10.1128/JCM.44.1.206-213.2006.
A multitarget real-time PCR (MRT-PCR) for detection of Chlamydia trachomatis DNA was developed and validated. There were three targets for amplification in a single reaction: the cryptic plasmid (CP), the major outer membrane protein (MOMP) gene, and an internal control. The assay had the following characteristics: (i) detection and confirmation of the presence of C. trachomatis DNA in a single reaction, (ii) detection of all genovars of C. trachomatis without any cross-reactivity with pathogenic bacteria or commensal organisms of the oropharynx and genital tract, (iii) a 95% probability of detection with three copies of MOMP and one copy of CP per reaction mixture, (iv) identification of the inhibition of amplification, (v) a quantitative dynamic range of 25 to 250,000 genome copies per reaction mixture, (vi) high intra- and interassay reproducibilities, and (vii) correct identification of all samples in the validation panel. There were 146 COBAS Amplicor PCR (Amplicor PCR)-positive samples and 122 Amplicor PCR-negative samples in the panel. MRT-PCR detected CP DNA alone in 6 (4%) Amplicor PCR-positive samples and both CP and MOMP DNAs in 140 (96%) of 146 Amplicor PCR-positive samples. The quantity of MOMP DNA in 95 (68%) of 140 samples was within the dynamic range of the assay. The median C. trachomatis load in these samples was 321 genome copies per reaction mixture (range, 26 to 40,137 genome copies per reaction mixture). Due to the inclusion of two different C. trachomatis-specific targets, the assay confirmed 259 (97%) of 268 results in a single reaction. This assay could be used in the qualitative format for the routine detection of C. trachomatis and in the quantitative format for study of the pathogenesis of C. trachomatis-associated diseases. The assay demonstrated the potential to eliminate the need for confirmatory testing in almost all samples, thus reducing the turnaround time and the workload.
开发并验证了一种用于检测沙眼衣原体DNA的多靶点实时PCR（MRT-PCR）方法。在单一反应中有三个扩增靶点：隐蔽质粒（CP）、主要外膜蛋白（MOMP）基因和一个内部对照。该检测方法具有以下特点：（i）在单一反应中检测并确认沙眼衣原体DNA的存在；（ii）检测所有沙眼衣原体基因变种，且与口咽部和生殖道的病原菌或共生生物无任何交叉反应；（iii）每个反应混合物中有三个拷贝的MOMP和一个拷贝的CP时，检测概率为95%；（iv）识别扩增抑制；（v）每个反应混合物的定量动态范围为25至250,000个基因组拷贝；（vi）检测内和检测间的重复性高；（vii）正确识别验证组中的所有样本。该验证组中有146个COBAS Amplicor PCR（Amplicor PCR）阳性样本和122个Amplicor PCR阴性样本。MRT-PCR在6个（4%）Amplicor PCR阳性样本中仅检测到CP DNA，在146个Amplicor PCR阳性样本中的140个（96%）中检测到CP和MOMP DNA。140个样本中的95个（68%）的MOMP DNA数量在检测的动态范围内。这些样本中沙眼衣原体载量的中位数为每个反应混合物321个基因组拷贝（范围为每个反应混合物26至40,137个基因组拷贝）。由于包含两个不同的沙眼衣原体特异性靶点，该检测方法在单一反应中确认了268个结果中的259个（97%）。该检测方法可用于沙眼衣原体常规检测的定性形式，以及沙眼衣原体相关疾病发病机制研究的定量形式。该检测方法显示出几乎消除所有样本中确证检测需求的潜力，从而减少周转时间和工作量。
