Institute of Virology, University of Bonn Medical Centre, 53127 Bonn, Germany.
Altona Diagnostics GmbH, Mörkenstrasse 12, 22767 Hamburg, Germany.
J Clin Virol. 2014 Jun;60(2):168-71. doi: 10.1016/j.jcv.2014.03.012. Epub 2014 Mar 28.
A highly pathogenic human coronavirus causing respiratory disease emerged in the Middle East region in 2012. In-house molecular diagnostic methods for this virus termed Middle East respiratory syndrome coronavirus (MERS-CoV) allowed sensitive MERS-CoV RNA detection in patient samples. Fast diagnosis is important to manage human cases and trace possible contacts.
The aim of this study was to improve the availability of existing nucleic acid amplification-based diagnostic methods for MERS-CoV infections by providing a real-time RT-PCR kit, including an internal control and two target regions recommended by the World Health Organization (WHO). And to validate this kit (RealStar MERS-CoV RT-PCR kit 1.0, Altona Diagnostics GmbH, Hamburg, Germany) using clinical samples of one MERS-CoV case from Munich and respiratory samples of patients with other respiratory diseases.
An internal amplification control was included into the RT-PCR assays targeting the genomic region upstream of the Envelope gene (upE) and within open reading frame (ORF) 1A. Based on these assays, a ready-to-use real-time RT-PCR kit featuring both the upE and ORF1A assays was developed, validated and compared to the established in-house versions.
The performance of both RT-PCR assays included in the kit is comparable to the in-house assays. They show high analytical sensitivity (upE: 5.3 copies/reaction; ORF1A: 9.3 copies/reaction), no cross-reactivity with other respiratory pathogens and detected MERS-CoV RNA in patient samples in almost the same manner as the in-house versions.
The kit is a valuable tool for assisting in the rapid diagnosis, patient management and epidemiology of suspected MERS-CoV cases.
一种高致病性的人类冠状病毒于 2012 年在中东地区引发呼吸道疾病。针对该病毒的内部分子诊断方法被称为中东呼吸综合征冠状病毒（MERS-CoV），可实现对患者样本中 MERS-CoV RNA 的灵敏检测。快速诊断对于管理人类病例和追踪可能的接触者至关重要。
本研究旨在通过提供一种基于核酸扩增的实时 RT-PCR 试剂盒，包括世界卫生组织（WHO）推荐的内部对照和两个靶标区域，改进现有的 MERS-CoV 感染核酸扩增诊断方法的可用性。该试剂盒（Altona Diagnostics GmbH，汉堡，德国的 RealStar MERS-CoV RT-PCR 试剂盒 1.0）使用慕尼黑的一例 MERS-CoV 病例的临床样本和其他呼吸道疾病患者的呼吸道样本进行验证。
针对包膜基因（upE）上游和开放阅读框（ORF）1A 内的基因组区域设计了内部扩增对照，基于这些检测，开发、验证了一种即用型实时 RT-PCR 试剂盒，包含 upE 和 ORF1A 检测。
试剂盒中包含的两种 RT-PCR 检测方法的性能与内部检测方法相当。它们具有高分析灵敏度（upE：5.3 拷贝/反应；ORF1A：9.3 拷贝/反应），与其他呼吸道病原体无交叉反应，并且以与内部版本几乎相同的方式检测到患者样本中的 MERS-CoV RNA。
该试剂盒是协助快速诊断、患者管理和疑似 MERS-CoV 病例流行病学研究的有价值工具。
