芋花叶病毒RNA提取前处理方法优化及快速检测体系的构建  

Optimization of Pretreatment Methods for RNA Extraction of DSMV and Construction of Rapid Detection Systems

作　　者：何玉书 吴玲玲 李欣霞 曾颖 张亚楠 HE Yu-shu;WU Ling-ling;LI Xin-xia;ZENG Ying;ZHANG Ya-nan(Xiamen Medical College,Xiamen 361023,China)

机构地区：[1]厦门医学院,福建厦门361023

出　　处：《特产研究》2022年第4期110-115,120,共7页Special Wild Economic Animal and Plant Research

基　　金：厦门医学院大学生创新创业训练计划资助项目(S202112631032X);厦门医学院大学生创新创业训练计划资助项目(202012631033)。

摘　　要：芋花叶病毒(Dasheen mosaic virus, DSMV)是芋在无性繁殖过程中不断积累并遗传的最为严重的一种植物病毒。为解决芋因受病毒侵染而导致的品种退化,构建快速且高效的芋花叶病毒RNA提取前处理及快速检测方法,本研究通过对芋叶总RNA提取前处理过程进行改良和优化,探究总RNA中DSMV的两步式RT-PCR、一步式RT-PCR及RT-qPCR快速检测体系。采用液氮联合组织研磨仪的植物总RNA提取前处理法,研磨时间20 min,研磨频率30(1/s),钢珠1个的最佳研磨条件,能获得含量纯度均较高的芋叶总RNA;一步式RT-PCR体系为总RNA模板1.0μL,10×PCR buffer 2.5μL,25 mM Mg1.0μL,2.5 mM dNTPs 1.0μL,10μmol/L上下游引物各1.0μL,200 U/μL逆转录酶0.5μL,5 U/μL Taq DNA聚合酶1.0μL,25.0μL总体系。一步式RT-PCR体系具有比两步式更快速、简便且灵敏的检测效果。Dasheen mosaic virus(DSMV) is the most serious plant virus that accumulates and inherits from taro during asexual reproduction.In order to solve the variety degradation of taro due to virus infection, and to construct an efficient and rapid method for pre-extraction and rapid detection of taro mosaic virus RNA, this study improved and optimized the pre-extraction process of total RNA from taro leaves. Twostep RT-PCR, one-step RT-PCR and RT-qPCR rapid detection systems for DSMV in total RNA were used, and the samples that were not detected by RT-PCR were re-examined by fluorescence quantitative PCR technology. The plant total RNA extraction method using liquid nitrogen combined with tissue grinder, grinding time of 20 min, grinding frequency of 30(1/s), and the optimal grinding conditions of one steel ball can obtain the total RNA of taro leaves with high content and purity. One-step RT-PCR system: 1.0μL of total RNA template, 2.5μL of 10×PCR buffer, 1.0μL of 25 mM Mg, 1.0μL of 2.5 mM dNTPs, 1.0μL of 10μmol/L upstream and downstream primers, 200 U/μL of reverse transcriptase 0.5μL, 5 U/μL Taq DNA Polymerase 1.0μL, 25.0μL system.The one-step RT-PCR system has a faster, simpler and more sensitive detection effect than the two-step method.

关 键 词：芋花叶病毒 RNA提取前处理 正交优化 一步式RT-PCR 

分 类 号：Q503[生物学—生物化学]