A multiplex PCR assay for the simultaneous detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis
A multiplex PCR assay for the simultaneous detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis
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DOI：
10.1016/j.yexmp.2015.01.011
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发表时间：
2015-04-01
影响因子：
3.6
通讯作者：
Tsongalis, Gregory J.
中科院分区：
文献类型：
--
作者：
Abou Tayoun, Ahmad N.;Burchard, Paul R.;Tsongalis, Gregory J.
导言：在发展中国家，性传播感染(STI)及其并发症被列为成年人求医的前五大疾病类别。沙眼衣原体(CT)、淋球菌(NG)和阴道毛滴虫(TV)是世界上最常见的三种性传播疾病，其中TV占一半以上。在发展中国家，诊断性传播感染的传统方法费时费力，往往不太敏感，而且周转时间很长，最新的商业诊断测试一次只针对一种或至多两种性传播感染。在这里，我们描述了一种高灵敏、快速和廉价的基于样本到答案的多重PCR方法，用于同时检测阴道毛滴虫、淋球菌和沙眼衣原体。材料和方法：我们设计了一种多重PCR方法，利用熔融曲线分析方法检测CT、TV、NG和过程/PCR对照4种靶标。为了确定每个病原体的检测限(LOD)，我们使用了先前提取并量化的TV、NG和CT基因组DNA(Vircell，西班牙)。对于每个目标，LOD是通过降低其拷贝数，同时逐步增加其他两个STI负载来确定的。在优化的方法中，过程/PCR控制保持不变，并在提取前添加到每个样品中。在一项一致性研究中，我们检测了26名患者的尿液、阴道和直肠拭子样本，这些样本对一种或多种测试的性传播感染呈阳性。此外，56份液体细胞学标本(ThinPrep)用于评估特异性。结果：该方法的循环时间不到2小时，在其他2个靶点存在的情况下，每个STI的检测限低至7-31个拷贝。我们的方法与26份已知临床标本的尿液、阴道和直肠拭子标本的符合率也达到了100%。TV、NG、CT和我们的过程/PCR对照分别一致地被鉴定为78摄氏度、823摄氏度、85.7摄氏度和类似92摄氏度。当应用于从残留的ThinPrep样本中提取的DNA时，54/56个样本的检测结果为阴性。两个样本被发现同时感染了CT。结论：我们的多重检测结合了一种快速且经济有效的分子诊断方法，以及在各种实验室环境下使用所需的多功能性。这些性能特点使这种多重STI检测非常适合在临床实验室使用。(C)2015 Elsevier Inc.保留所有权利。
Introduction: For developing countries, sexually transmitted infections (STIs) and their complications are ranked in the top 5 disease categories for which adults seek medical treatment. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV) are the three most common STIs worldwide, with TV accounting for over half of the cases. In developing countries, traditional methods for diagnosing STIs are laborious, often not very sensitive, and have a long turnaround time with most recent commercially available diagnostic tests targeting one or, at most, two of these STIs at a time. Here, we describe the development of a highly sensitive, rapid and affordable sample-to-answer multiplex PCR-based assay for the simultaneous detection of Trichomonas vaginalis, Neisseria gonorrhoeae, and Chlamydia trachomatis.Materials and Methods: We designed a multiplex PCR assay for the detection of 4 targets (CT, TV, NG, and process/PCR control) using melt curve analysis. To establish the limit of detection (LOD) for each pathogen, we used previously extracted and quantified TV, NG, and CT genomic DNA (Vircell, Spain). For each target, the LOD was determined by lowering its copy number while increasing the other two STI loads in a stepwise fashion. The process/PCR control remained constant in the optimized assay and was spiked into each sample before extraction. For a concordance study, we tested urine, vaginal and rectal swab specimens from 26 patients positive for one or more of the tested STIs. In addition, 56 liquid cytology specimens (Thinprep) were used to assess specificity.Results: This assay has a turnaround time of less than 2 h and has a limit of detection as low as 7-31 copies for each STI in the presence of the other 2 targets. Our assay also demonstrated 100% concordance with 26 known clinical samples from urine, vaginal and rectal swab specimens. TV, NG, CT, and our process/PCR control were consistently identified at 78 degrees C, 823 degrees C, 85.7 degrees C, and similar to 92 degrees C, respectively. When applied to DNA extracted from residual Thinprep specimens, the assay was negative in 54/56 samples. Two samples were found to be co-infected with CT.Conclusions: Our multiplex assay combines a rapid and cost-effective approach to molecular diagnostics with the versatility required for use within a variety of laboratory settings. These performance characteristics make this multiplex STI assay highly suitable for use in a clinical laboratory. (C) 2015 Elsevier Inc. All rights reserved.