马铃薯帚顶病毒PMTV三重RT-PCR检测体系的建立及应用

Establishment and application of triple RT-PCR detection method for potato mop-top virus

摘要

马铃薯帚顶病毒（potato mop-top virus，PMTV）是马铃薯重要的检疫性病毒，严重危害马铃薯的产量和品质。为高效、灵敏地检测马铃薯中的 PMTV，本研究基于 PMTV 编码复制（RNA1）、外壳蛋白（RNA2）和移动（RNA3）的三条独立基因组 RNA，针对 RNA1 设计特异性引物，筛选 RNA2 与 RNA3 已发表引物，并优化反应条件，成功构建 PMTV 的三重逆转录聚合酶链式反应即 RT-PCR 检测体系。该检测体系对 PMTV 具有高灵敏度和强特异性，最低检测限为 0.1 ng·μL^-1 cDNA，且可同时检测到 RNA1（265 bp）、RNA2（460 bp）和 RNA3（769 bp）3 段目的基因，无交叉反应。利用该检测体系对来自云南、广东、贵州等地的 200 份马铃薯样品进行检测，共发现 24 份阳性样本，阳性率为 12%。该体系可稳定检测不同组织类型（叶片、试管苗、块茎）样品，适用于田间样本快速筛查与种薯质量监测。此外，基于 9 份阳性样本的 3 段目的基因序列构建的系统进化树显示，我国 PMTV 分离株分布于多个进化分支，提示其可能存在不同来源或本地适应性演化。本研究建立的三重 RT-PCR 检测体系为 PMTV 的快速检测、分子流行病学调查及种薯健康认证提供了可靠技术手段。

Abstract

Potato mop-top virus（PMTV）is an important quarantine virus of potato, causing severe damage to both yield and quality. For efficient and sensitive detection of PMTV in potato, this study developed a triple RT-PCR assay based on the three independent genomic RNAs of PMTV, which encode the replication（RNA1）, coat protein（RNA2）, and movement（RNA3）. Specifically, specific primers were designed targeting RNA1, while previously published primers for RNA2 and RNA3 were screened. Following optimization of the reaction conditions, a triple reverse transcription polymerase chain reaction（RT-PCR）detection system for PMTV was successfully established. The developed assay exhibited high sensitivity and strong specificity for PMTV detection, with a detection limit of 0.1 ng · μL^-1 cDNA. It successfully and simultaneously amplified the three target genes: RNA1（265 bp）, RNA2（460 bp）, and RNA3（769 bp）, without any cross-reactivity. Using this detection system, a total of 200 potato samples collected from Yunnan, Guangdong, Guizhou and other regions were tested, revealing 24 positive samples with a positive rate of 12%. The developed assay demonstrated reliable detection across various tissue types, including leaves, in vitro plantlets, and tubers, making it suitable for rapid field screening and seed potato quality monitoring. Furthermore, phylogenetic analysis based on three target nucleotide sequences of nine positive samples revealed that Chinese PMTV isolates were distributed across multiple evolutionary clades, suggesting possible diverse origins or local adaptive evolution. The triplex RT-PCR detection system established in this study provides a reliable technical tool for rapid detection of PMTV, molecular epidemiological surveillance, and seed potato health certification.

关键词

马铃薯 / 帚顶病毒 / 三重 / RT-PCR 检测

Key words

Potato / PMTV / Triple / RT-PCR detection

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基金资助

齐齐哈尔市科技计划联合引导项目(LNYGG-2024006)

黑龙江省农业科技创新跨越工程农业科技基础创新优青项目(CX22YQ31)

齐齐哈尔市科学技术计划重点项目(ZDGG-202210)

黑龙江省农业科技创新跨越工程农业关键技术科技创新重点攻关项目(CX23GG11)

科技部、财政部国家科技资源共享服务平台项目(NCGRC-2025-44)

财政部和农业农村部国家现代农业产业技术体系(CARS-09-P16)