马铃薯卷叶病毒PLRV RT-LAMP检测方法优化

Optimization of the PLRV RT-LAMP detection method

马铃薯卷叶病毒Potato leafroll virus(PLRV)是目前严重影响马铃薯产量与品质的主要病毒之一,给马铃薯产业造成巨大损失。本研究采用环介导等温核酸扩增(loop-mediated isothermal amplification, LAMP)技术建立PLRV的RT-LAMP检测方法。采取单因素变化试验,对RT-LAMP反应体系中多个因素包括引物组合、温度条件及Mg2+、betaine、Bst 3.0 DNA聚合酶、dNTPs、UNG、SYBR GreenⅠ和引物组合的浓度进行一系列试验和优化。采用RT-PCR检测方法进行平行比对试验,对优化后的RT-LAMP反应体系进行了验证。结果表明,最佳引物组合为P3,最适反应温度62℃,25μL反应体系中,Mg2+、betaine、Bst 3.0 DNA聚合酶和UNG的最佳终浓度分别为4 mmol/L、0 mmol/L、0.64 U/μL和0.08 U/μL,dNTPs的最佳用量为1μL(dATP、dGTP、dCTP各0.4 mmol/L,dUTP 1.2 mmol/L),SYBR GreenⅠ(20×)的最佳用量1μL,primer mix的最佳用量2.5μL(PLRV-FIP/BIP、PLRV-F3/B3和PLRV-LF/LB的浓度分别为0.8、0.2μmol/L和0.6μmol/L),RNA模板1μL(2 ng/μL),加DEPC-H2O至25μL,反应时间50 min。优化后的RT-LAMP检测结果与RT-PCR一致,且可视化判读结果。因此,建立的PLRV RT-LAMP检测方法为进一步开发RT-LAMP检测试剂盒及其实际应用奠定了基础。

Potato leafroll virus(PLRV) is currently one of the main threats for the yield and quality of potatoes, having caused tremendous damages to the potato industry. In this study, a PLRV reverse transcription-loop mediated isothermal amplification(RT-LAMP) method was established based on the loop-mediated isothermal amplification(LAMP). Single-factor experiments were conducted to test and optimize the RT-LAMP reaction system, including the primers, temperature, Mg2+, betaine, Bst 3.0 DNA polymerase, dNTPs, UNG, SYBR Green Ⅰ and primer mix concentrations. The optimized RT-LAMP reaction system was then verified through parallel-controlled test using the reverse transcription-polymerase chain reaction(RT-PCR) method. The results showed that, in the optimized reaction conditions, the primer pair was P3 and the reaction temperature was 62℃;in the 25 μL reaction system, the concentrations of Mg2+, betaine Bst 3.0 DNA polymerase, and UNG were 4 mmol/L, 0 mmol/L, 0.64 U/μL, and 0.08 U/μL, respectively;the dosage for dNTPs was 1 μL(0.4 mmol/L for dATP, dGTP, and dCTP, respectively, and 1.2 mmol/L for dUTP);the dosage for SYBR Green Ⅰ(20×) was 1 μL;the dosage for the primer mix was 2.5 μL(the corresponding concentrations for PLRV-FIP/BIP, PLRV-F3/B3, and PLRV-LF/LB was 0.8 μmol/L, 0.2 μmol/L, and 0.6 μmol/L, respectively);for the 1 μL RNA template(2 ng/μL), additional DEPC-H2O was added to get a final 25 μL volume, and the reaction time was 50 min. The optimized RT-LAMP provided the same detection result as RT-PCR and the interpretation could be visualized. These results confirmed that our PLRV RT-LAMP reaction system provides a basis for further developing RT-LAMP detection kits and for their practical application.