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【目的】建立一种利用颜色判定的快速、简单、灵敏度高的检测方法,即可视化的逆转录环介导等温扩增(RT-LAMP)方法,并应用于人A组轮状病毒的基因检测。【方法】针对A组轮状病毒VP6基因的6个保守区域设计4条特异性引物,在64°C恒温条件下进行核酸扩增反应1 h,在扩增前加入染料钙黄绿素(Calcein)作为反应指示剂,以钙黄绿素的颜色变化作为结果判定标准。评价该方法的特异性和灵敏度,并同时利用RT-LAMP和RT-PCR两种方法对90份临床腹泻样本中的病毒核酸进行检测。【结果】RT-LAMP方法特异性较高,灵敏度可以达到103 copies/μL RNA分子水平,比RT-PCR高出100倍。对临床标本的检出率与RT-PCR方法相当。【结论】建立的RT-LAMP法灵敏度较高,特异性强,节省时间,结果可视化,具有野外检测和现场快速检测的潜力。
【OBJECTIVE】 To establish a rapid, simple and sensitive detection method using colorimetric determination, that is visual RT-LAMP method and applied to the genetic testing of human A group rotavirus . 【Method】 Four specific primers were designed according to the six conserved regions of VP6 gene of group A rotavirus. The amplification reaction was carried out at 64 ° C for 1 h, and the dye Calcein was added before amplification Response indicator, the color change of calcein as a result of the decision criteria. The specificity and sensitivity of this method were evaluated and the viral nucleic acids in 90 clinical samples of diarrhea were tested both by RT-LAMP and RT-PCR. 【Result】 The RT-LAMP method was highly specific and sensitive to 103 copies / μL RNA, which was 100 times higher than that of RT-PCR. The detection rate of clinical specimens and RT-PCR method is quite. 【Conclusion】 The established RT-LAMP method has the advantages of high sensitivity, strong specificity, time saving and visualization of results. It has the potential of field detection and rapid on-site detection.