Lau Lok Ting, Fung Yin-Wan Wendy, Wong Freda Pui-Fan, Lin Selma Sau-Wah, Wang Chen Ran, Li Hui Li, Dillon Natalie, Collins Richard A, Tam John Siu-Lun, Chan Paul K S, Wang Chen G, Yu Albert Cheung-Hoi
Key Laboratory of Neuroscience, Neuroscience Research Institute, Peking University and Department of Neurobiology, Peking University Health Science Center, 38 Xue Yuan Road, 100083, Beijing, China.
Biochem Biophys Res Commun. 2003 Dec 26;312(4):1290-6. doi: 10.1016/j.bbrc.2003.11.064.
An enhanced polymerase chain reaction (PCR) assay to detect the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) was developed in which a target gene pre-amplification step preceded TaqMan real-time fluorescent PCR. Clinical samples were collected from 120 patients diagnosed as suspected or probable SARS cases and analyzed by conventional PCR followed by agarose gel electrophoresis, conventional TaqMan real-time PCR, and our enhanced TaqMan real-time PCR assays. An amplicon of the size expected from SARS-CoV was obtained from 28/120 samples using the enhanced real-time PCR method. Conventional PCR and real-time PCR alone identified fewer SARS-CoV positive cases. Results were confirmed by viral culture in 3/28 cases. The limit of detection of the enhanced real-time PCR method was 10(2)-fold higher than the standard real-time PCR assay and 10(7)-fold higher than conventional PCR methods. The increased sensitivity of the assay may help control the spread of the disease during future SARS outbreaks.
开发了一种用于检测与严重急性呼吸综合征相关的冠状病毒（SARS-CoV）的增强型聚合酶链反应（PCR）检测方法，其中在TaqMan实时荧光PCR之前进行靶基因预扩增步骤。从120例被诊断为疑似或可能的SARS病例的患者中收集临床样本，并通过常规PCR随后进行琼脂糖凝胶电泳、常规TaqMan实时PCR和我们的增强型TaqMan实时PCR检测方法进行分析。使用增强型实时PCR方法从120份样本中的28份获得了预期大小的SARS-CoV扩增子。单独的常规PCR和实时PCR鉴定出的SARS-CoV阳性病例较少。在28例中的3例中通过病毒培养证实了结果。增强型实时PCR方法的检测限比标准实时PCR检测高100倍，比常规PCR方法高10000000倍。该检测方法灵敏度的提高可能有助于在未来SARS疫情爆发期间控制疾病的传播。
