柑橘衰退病毒RT-LAMP快速检测方法的建立

A RT-LAMP Assay for Detection of Citrus Tristeza Virus

【目的】建立一种反转录环介导等温扩增(reverse transcription loop-mediated isothermalamplification,RT-LAMP)检测柑橘衰退病毒(Ctrus tristeza virus,CTV)的一步法检测技术。【方法】根据CTV保守的p25设计2对特异性引物,对RT-LAMP体系的最佳反应条件、特异性、灵敏度等进行研究。【结果】建立了一种特异性检测CTV的RT-LAMP方法,该方法的灵敏度比RT-PCR方法高100倍,产物通过Acc I酶切得到验证,而且反应产物中加入SYBR Green I可直接观察样品是否感染CTV。【结论】RT-LAMP法检测CTV,其成本低、准确且快速,可满足基层、科研单位等对该病害检测的需要。

[ Objective ] The objective of this study is to establish a quick and accurate detection technique of Citrus tristeza virus by reverse transcription loop-mediated isothermal amplification （RT-LAMP）. [Method] According to published GenBank sequences, two pairs of primers were designed in the conserved region of CTVp25. The concentration of primers, reaction time and other conditions of RT-LAMP were optimized in order to improve specificity and sensitivity. [Result] A rapid and specific RT-LAMP method for detection of CTV was established. Sensitivity of the RT-LAMP assay was 100-fold higher than a standard RT-PCR method. The RT-LAMP products were confirmed by digestion with Acc I restriction enzyme. Products amplified by RT-LAMP were visible to add SYBR Green I, suggesting that it can be used to detect CTV directly. [Conclusion] The detection process can be speeded up by using RT-LAMP under routine conditions at low cost and high accuracy with simple facilities and can be used as an excellent method for CTV detection in both research institutions and rural areas.