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目的应用环介导等温扩增(LAMP)技术建立SARS冠状病毒可视化快速检测方法。方法针对SARS冠状病毒RNA聚合酶编码基因保守区设计LAMP引物及环引物,反应体系加入钙黄绿素作为LAMP扩增反应指示剂,结合LAMP浊度仪优化扩增条件,根据钙黄绿素的颜色变化进行结果判定,评价所建立LAMP方法的特异性和灵敏性。结果本方法最低检出限约为10拷贝/反应管,高于普通PCR;检测特异性高,仅SARS冠状病毒反应管钙黄绿素颜色由褐色变为黄绿色,而甲型H1N1病毒、高致病性H5亚型禽流感病毒、普通流感病毒均不发生变色反应;体系的扩增效率高于普通PCR,35 min内出结果。结论所建立的基于颜色判定的SARS病毒检测方法具有特异、灵敏、设备要求简单等特点,适用于SARS冠状病毒现场快速检测。
Objective To establish rapid visualization of SARS-CoV by ring-mediated isothermal amplification (LAMP) technique. Methods LAMP primers and loop primers were designed according to conserved regions of RNA polymerase coding RNA of SARS coronavirus. Calcein was added into the reaction system as an indicator of LAMP amplification reaction. LAMP turbidimeter was used to optimize the amplification conditions. The results were based on the color change of calcein To determine and evaluate the specificity and sensitivity of the established LAMP method. Results The detection limit of this method was about 10 copies / reaction tube, which was higher than that of normal PCR. The detection specificity was high. Only the color of calcein of SARS coronavirus changed from brown to yellowish green, while the type A H1N1 virus, H5 subtype avian influenza virus and common influenza virus did not change color reaction; the amplification efficiency of the system was higher than that of ordinary PCR, and the result was within 35 min. Conclusion The SARS virus detection method based on color judgment established is characterized by its specificity, sensitivity and simple equipment requirements. It is suitable for the rapid detection of SARS coronavirus in the field.