A组轮状病毒可视化RT-LAMP方法的建立

Development of RT-LAMP for rapid visual detection of group A rotavirus

【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] A rapid, simple and sensitive visual reverse transcription loop-mediated isothermal amplification method was established to detect group A rotavirus. [Methods] The method employed a set of four specially designed primers that recognize six distinct sequences of the VP6 gene for amplification of nucleic acid under isothermal conditions at 64 ℃ for one hour. The amplification process of RT-LAMP was monitored by the addition of Calcein dye prior to amplification. The specificity and sensitivity of the RT-LAMP assay was assessed and the assay was further evaluated with 90 clinical specimens of diarrhea patients with RT-LAMP and RT-PCR detection. [Results] The results showed that the RT-LAMP was able to achieve a sensitivity of 10^3 copies/μL with a high specificity. The detection limit of RT-LAMP was 100-fold higher than that of RT-PCR. Positive rate of RT-LAMP in analysis of clinical specimens was approximately the same as that of RT-PCR as well. [Conclusion] The RT-LAMP assay has been proven to be a rapid, sensitive, specific and visual method for detection of the group A rotavirus, and that the RT-LAMP assay is potentially useful for the field detection and rapid detection on spot.