Li Guan, Nie Kai, Zhang Dan, Li Xinna, Wang Yanqun, Tan Wenjie, Ma Xuejun
Bing Du Xue Bao. 2015 May;31(3):269-75.
A simple, rapid and sensitive colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) was developed for rapid detection of Middle East respiratory syndrome coronavirus (MERS-CoV). The method employed six primers that recognized sequences of a nucleocapsid gene for amplification of nucleic acids under isothermal conditions at 63 degrees C for 60 min. Products were detected through a LA-320c Loopamp Turbidimeter (real-time RT-LAMP) or visual inspection of color change by pre-addition of Hydroxynaphthol Blue dye (visual RT-LAMP). Specificity of RT-LAMP was validated by detection of several human coronaviruses and common respiratory viruses. MERS-CoV real-time RT-LAMP had a linear correlation (R2) of 0.995 at 10(3)-10(6) copies. The limit of detection of real-time RT-LAMP, visual RT-LAMP and quantitative real-time PCR was 500, 1000 and 100 copies/reaction, respectively. The established RT-LAMP assay was demonstrated to be a rapid screening tool for MERS-CoV infection, and could be suitable in resource-limited clinical sites and for field studies.
为快速检测中东呼吸综合征冠状病毒（MERS-CoV），开发了一种简单、快速且灵敏的比色逆转录环介导等温扩增（RT-LAMP）方法。该方法使用六条引物识别核衣壳基因序列，在63℃等温条件下扩增核酸60分钟。产物通过LA-320c环介导等温扩增浊度仪（实时RT-LAMP）检测，或通过预先添加羟基萘酚蓝染料进行颜色变化的目视检查（目视RT-LAMP）。通过检测几种人类冠状病毒和常见呼吸道病毒验证了RT-LAMP的特异性。MERS-CoV实时RT-LAMP在10³-10⁶拷贝数时线性相关系数（R²）为0.995。实时RT-LAMP、目视RT-LAMP和定量实时PCR的检测限分别为500、1000和100拷贝/反应。所建立的RT-LAMP检测方法被证明是一种用于MERS-CoV感染的快速筛查工具，适用于资源有限的临床场所和现场研究。
