摘要
建立中东呼吸系统综合征冠状病毒(MERS-CoV)感染的筛查与诊断应用的核酸检测方法。本研究建立了基于MERS-CoV三个分子靶标(upE,ORF1b,N2)的双重以及三重的三种荧光定量RT-PCR检测技术方法,分别与前期建立的单重荧光定量RT-PCR(upE或N2)检测方法做了比较,并且采用MERS-CoV病毒毒株、上海提供的口岸发烧病人样本以及由首例中国MERS-CoV韩国地区输入病例的咽拭子样品和全血的样品做了临床验证。结果显示:采用MERS-CoV病毒毒株作为模板,三种新建立的多重检测方法与单重荧光定量RT-PCR检测方法最低检测限均能达到10PFU/mL,且与其他常见呼吸道病毒的阳性样本均无交叉反应,特异性良好。对临床样品的验证中,上海病人咽拭子样本均为阴性,而中国首例MERS输入病例的急性期咽拭子样品均为阳性,而全血样的检测结果显示N2靶标有较高检出率,明显优于基于upE单一靶标。本研究所建立的基于MERS-CoV三个分子靶标(upE,ORF1b,N2)的双重以及三重荧光定量RT-PCR检测技术方法用于MERS-CoV感染的实验室诊断,具有较高灵敏度与特异性及适用性。
We aimed to develop a novel laboratory assay for the detection of Middle East respiratory syndrome coronavirus（MERS-CoV）infection.Several novel multiplex real-time RT-PCR assays were developed using the upE,ORF1 band N2genes of MERS-CoV as targets;the novel assays were compared with previous monoplex real-time RT-PCR assays.For validation,we tested a MERS-CoV strain（hCoVEMC）,clinical specimens from patients with fever in Shanghai,and specimens from the first imported MERS case in China.The detection limit of the novel multiplex real-time RT-PCR assays was 10 PFU of MERS-CoV per ml,the same as that in monoplex real-time RT-PCR assays based on upE or N2.The detection was specific for MERS-CoV.In validation using clinical samples,pharyngeal swabs from Shanghai patients were detected as negative,while swabs from the first imported MERS case in China were detected as positive.Using whole blood samples from a MERS case,better detection results were obtained with N2 as the target than upE.We conclude that all the novel assays established in this study could be used for the detection of MERS-CoV;they show potential for improvement compared with monoplex real-time RT-PCR assay based on upE.
出处
《病毒学报》
CAS
CSCD
北大核心
2016年第3期349-354,共6页
Chinese Journal of Virology
基金
传染病重大专项(2014ZX10004-001
201310004-101
2013ZX10004601)
