Smits Saskia L, Raj V Stalin, Pas Suzan D, Reusken Chantal B E M, Mohran Khaled, Farag Elmoubasher A B A, Al-Romaihi Hamad E, AlHajri Mohd M, Haagmans Bart L, Koopmans Marion P
Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands; ViroClinics BioSciences BV, Marconistraat 16, 3029 AK Rotterdam, Netherlands.
Department of Viroscience, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, Netherlands.
J Clin Virol. 2015 Mar;64:83-7. doi: 10.1016/j.jcv.2014.12.006. Epub 2014 Dec 15.
Middle East Respiratory Syndrome coronavirus (MERS-CoV) is an emerging pathogen that causes lower respiratory tract infection in humans. Camels are the likely animal source for zoonotic infection, although exact transmission modes remain to be determined. Human-to-human transmission occurs sporadically. The wide geographic distribution of MERS-CoV among dromedary camels and ongoing transmissions to humans provides concern for the evolution of a MERS-CoV variant with efficient human-to-human transmission capabilities. Phylogenetic analysis of MERS-CoV has occurred by analysis of full-length genomes or multiple concatenated genome fragments, which is time-consuming, costly and limited to high viral load samples.
To develop a simple, reliable MERS-CoV variant typing assay to facilitate monitoring of MERS-CoV diversity in animals and humans.
Phylogenetic analysis of presently known full-length MERS-CoV genomes was performed to identify genomic regions with sufficient phylogenetic content to allow reliable MERS-CoV variant typing. RT-PCR assays targeting these regions were designed and optimized.
A reverse-transcription PCR assay for MERS-CoV targeting a 615 bp spike fragment provides a phylogenetic clustering of MERS-CoV variants comparable to that of full-length genomes. The detection limit corresponds to a cycle treshold value of ∼ 35 with standard upE real time PCR assays on RNA isolated from MERS-CoV EMC. Nasal swabs from RT-PCR positive camels (Ct values 12.9-32.2) yielded reliable sequence information in 14 samples.
We developed a simple, reliable MERS-CoV variant typing assay which is crucial in monitoring MERS-CoV circulation in real time with relatively little investment on location.
中东呼吸综合征冠状病毒（MERS-CoV）是一种新出现的病原体，可导致人类下呼吸道感染。骆驼可能是这种人畜共患病感染的动物源，尽管确切的传播方式仍有待确定。人传人现象偶尔发生。MERS-CoV在单峰骆驼中的广泛地理分布以及持续向人类的传播引发了人们对具有高效人传人能力的MERS-CoV变异株进化的担忧。对MERS-CoV的系统发育分析是通过对全长基因组或多个串联的基因组片段进行分析来进行的，这既耗时又昂贵，并且仅限于高病毒载量样本。
开发一种简单、可靠的MERS-CoV变异株分型检测方法，以促进对动物和人类中MERS-CoV多样性的监测。
对目前已知的MERS-CoV全长基因组进行系统发育分析，以确定具有足够系统发育信息的基因组区域，从而实现可靠的MERS-CoV变异株分型。针对这些区域设计并优化了RT-PCR检测方法。
针对一个615 bp刺突片段的MERS-CoV逆转录PCR检测方法，其MERS-CoV变异株的系统发育聚类与全长基因组的聚类相当。检测限对应于使用标准upE实时PCR检测法对从MERS-CoV EMC分离的RNA进行检测时约为35的循环阈值。来自RT-PCR阳性骆驼（Ct值为12.9 - 32.2）的鼻拭子在14个样本中产生了可靠的序列信息。
我们开发了一种简单、可靠的MERS-CoV变异株分型检测方法，这对于以相对较少的现场投入实时监测MERS-CoV的传播至关重要。
