ICS 65.020 CCS B 16 湖 DB43 南 省 地 方 标 准 DB43/T 1 9 57—2020 稻曲病菌分子检测技术规程 Technical Regulations for Molecular Detection of Ustilaginoidea virens 2020-12-29发布 湖南省市场监督管理局 2021-03-29实施 发 布 DB43/T 1957—2020 目 次 前言························································································································ Ⅲ 1 范围 ····················································································································· 1 2 规范性引用文件 ······································································································ 1 3 术语和定义 ············································································································ 1 4 原理 ····················································································································· 1 5 仪器设备与试剂 ······································································································ 1 5.1 仪器设备 ········································································································· 1 5.2 主要试剂 ········································································································· 1 6 采样 ····················································································································· 2 6.1 样品采集 ········································································································· 2 6.2 样品贮运与存放 ································································································ 2 7 操作方法 ··············································································································· 2 7.1 样品 DNA 制备 ··································································································· 2 7.2 检测 ··············································································································· 2 8 结果判定 ··············································································································· 3 9 样品保存与处理 ······································································································ 3 附录 A（资料性） 稻曲病相关资料 ················································································ 4 附录 B（规范性） 水稻稻曲病菌鉴定报告 ······································································· 5 I DB43/T 1957—2020 II DB43/T 1957—2020 前 言 本文件按照 GB/T 1.1—2020 给出的规则起草。 请注意本文件的某些内容可能涉及专利。本文件的发布机构不承担识别这些专利的责任。 本文件由湖南省农业农村厅提出。 本文件由湖南省农业标准化技术委员会归口。 本文件起草单位：湖南省植物保护研究所、株洲市农业科学研究所。 本文件主要起草人：陈岳、谭新球、李成刚、吴龙云、王淋玉、张卓、张鑫、史晓斌、李小娟、肖 友伦、张德咏、刘勇。 III DB43/T 1957—2020 IV DB43/T 1957—2020 稻曲病菌分子检测技术规程 1 范围 本文件规定了稻曲病菌 PCR 检测的样品制备、检测技术和操作程序。 本文件适用于土壤、水稻种子和稻谷中稻曲病菌的分子检测。 2 规范性引用文件 下列文件中的内容通过文中的规范性引用而构成本文件必不可少的条款。其中，注日期的引用文件， 仅该日期对应的版本适用于本文件；不注日期的引用文件，其最新版本（包括所有的修改单）适用于本 文件。 GB/T 6682 分析实验室用水规格和试验方法 3 术语和定义 下列术语和定义适用于本文件。 稻曲病菌无性态为 Ustilaginoidea virens，属半知菌亚门、腔孢纲、瘤座孢目、绿核菌属，病原有性 阶段有性态为 Claviceps oryzae sativae，属子囊菌门、子囊菌纲、麦角菌属、稻麦角菌。稻曲病主要危 害水稻穗部并形成稻曲球，严重影响水稻的产量和稻米品质。见附录 A。 PCR（Polymerase Chain Reaction） ，聚合酶链式反应，是指在 DNA 聚合酶催化下，以母链 DNA 为模 板，以特定引物为延伸起点，通过变性、退火、延伸等步骤，体外复制出与母链模板 DNA 互补的子链 DNA 的过程。是一项 DNA 体外合成放大技术，能快速特异地在体外扩增任何目的 DNA。 4 原理 核糖体基因转录间隔区（Internal transcribed spacer ITS）进化速率较编码区快，在真菌种间存在着 丰富的变异，而在种内不同菌株间却高度保守，可以为病原菌的分子检测提供理想的靶序列。根据稻曲 病菌 ITS 的特异序列，合成 1 对特异性引物，获得稻曲病菌特异性扩增片段，用于土壤、水稻种子和稻 谷中稻曲病菌的快速分子检测。 5 仪器设备与试剂 5.1 仪器设备 PCR 检测仪、凝胶成像仪、土壤取样器、台式离心机、纯水仪、电泳仪、旋涡振荡器等。 5.2 主要试剂 1 DB43/T 1957—2020 苯酚/氯仿/异戊醇、乙醇、二甲基亚砜、氢氧化钠、脱脂奶粉、DNA marker、吐温 20、PCR 试剂盒 等。 除特别说明以外，本文件所用试剂均为分析纯，实验用水应符合 GB/T 6682 的规定。 6 采样 6.1 样品采集 6.1.1 土壤 水稻田 W 型 5 点取样，取 1-10 cm 深度的 50 g 土壤样品。 6.1.2 水稻种子和稻谷 将稻种或稻谷混匀后称取 50 g。 6.2 样品贮运及存放 样品采集后，放入密闭的容器内，送实验室。将取样结果进行记录，记录表格见附录 B。处理的样 品应保存在-20℃以下，避免反复冻融。 7 操作方法 7.1 样品 DNA 制备 7.1.1 土壤中 DNA 的提取 土壤风干后，制成 100 目的土壤样品，取 10 g 土壤加入 5 mL 无菌水，冷冻抽干 24-48 h，加入少 量石英砂，倒入液氮充分研磨，将研磨后的土壤分装至 1.5 mL 离心管中，每管加入 500 μl 0.4％脱 脂奶粉溶液，涡旋混匀。12000 rpm/min 离心 10 min。取上清加入等体积浓度为 20 μg/mL 的蛋白酶 K 缓冲液，55℃水浴 2h。水浴结束后，加入 1/2 体积的 7.5 M NH4Ac 溶液，混匀。12000 rpm/min 离心 10 min。吸上清液加入 2 倍体积无水乙醇，-20℃沉淀 0.5 h，12000 rpm/min 离心 10 min，去除上清液， 70％乙醇洗涤沉淀物，室温晾干。提取的 DNA 用 20μl 含 RNA 酶的 TE 缓冲液溶解，-20℃保存备用。 土壤 DNA 也可用土壤 DNA 提取试剂盒提取。 7.1.2 种子和稻谷中 DNA 的提取 将混匀后的稻种液氮研磨成粉末，称取 0.1 g 于 2 mL 管子中，加入 800 μl CTAB 提取液，65℃水 浴 15 min，期间反复摇匀；加入等体积苯酚/氯仿/异戊醇，混匀后摇床轻摇 20 min；12000 rpm/min 离 心 10 min，移上清 500 μl，加入 2 倍体积的无水乙醇，-20℃沉淀 0.5 h，12000 rpm/min 离心 10 min， 去除上清液，70％乙醇洗涤沉淀物，室温晾干。提取的 DNA 用 20μl 含 RNA 酶的 TE 缓冲液溶解，-20℃ 保存备用。 7.2 检测 7.2.1 PCR 检测 2 DB43/T 1957—2020 7.2.1.1 反