基于逆转录-环介导等温扩增技术快速检测发热伴血小板减少综合征病毒方法的建立

Development of A Reverse Transcription Loop-mediated Isothermal Amplification(RT-LAMP)assay for rapid detection of severe fever with thrombocytopenia syndrome virus

目的建立1种快速、特异、灵敏的发热伴血小板减少综合征病毒(SFTSV)的检测方法。方法针对SFTSV M基因保守区域的核酸序列,设计逆转录-环介导等温扩增(RT-LAMP)特异性引物,优化反应体系和条件。用毛细管电泳法和横向流动试纸条(LFD)法检测扩增产物,建立RT-LAMP检测方法。进行敏感性、特异性检验,并与Real-time RT-PCR法进行比较。结果以LFD法和毛细管电泳法检测RT-LAMP扩增产物具有相同的敏感性和特异性;RT-LAMP-LFD检测SFTSV的敏感性为10copies RNA分子/反应,且与其他病毒无交叉反应。RT-LAMP-LFD和Real-time RT-PCR检测临床标本阳性率差异无统计学意义(P>0.05),2种方法一致性好(Kappa=0.918)。结论建立的RT-LAMP方法快速、简单、操作简单,具有较高的敏感性和特异性,适合应用于基层单位和现场SFTSV的快速检测。

Objective To establish a rapid, specific and sensitive assay for the detection of severe fever with thrombocytopenia syndrome virus （SFTSV）. Methods The RT-LAMP primers were designed to target the conservative regions of the M segment of SFTSV. After optimization, the RT-LAMP products were simultaneously detected by capillary electrophoresis and lateral flow devices （LFD）. After assessing the sensitivity and specificity of the assay, the performance of the assay was com- pared with Real-time RT-PCR. Results With product detection, the LFD method showed a similar sensitivity and specificity as compared with capillary electrophoresis. The analytical sensitivity of the RT-LAMP-LFD assay was 10 copies of RNA per reaction. Furthermore, no cross-reaction was found when the assay was used to detect other related viruses. After evaluation with clinical specimens, RT-LAMP-LFD and Real-time RT-PCR were shown a high consistency （Kappa = 0. 918） and there was no significant difference in the detection rates of positive specimens （P〉0.05）. Conclusion The characteristics of rapidity, simplicity, high sensitivity and specificity, and low-cost make this RT-LAMP-LFD assay suitable for low-equipment setting laboratory and for on-site testing.