一步法 RT-PCR 检测李痘病毒及其实时定量 PCR 分析。

Detection of Prune dwarf virus by one-step RT-PCR and its quantitation by real-time PCR.

机构信息

Department of Virology, Crop Research Institute, Drnovska 507, 161 06 Prague 6, Czech Republic.

出版信息

J Virol Methods. 2010 Mar;164(1-2):139-44. doi: 10.1016/j.jviromet.2009.11.032. Epub 2009 Dec 7.

DOI:10.1016/j.jviromet.2009.11.032

PMID:20003914

Abstract

A quantitative PCR, real-time RT-PCR, and one-step real-time RT-PCR using SYBR Green-based tools for reliable detection and relative quantitation of Prune dwarf virus (PDV) in stone fruits are described. The assay reliability was tested on 55 samples from different hosts and regions. The sensitivity of the assay was also compared with other assays with different primers. Two plant-expressed genes, actin and 18S rRNA, were used as housekeeping genes for accurate quantitation of PDV in stone fruit trees. The expression of the gene for actin and the 18S ribosomal RNA gene corresponded with each other accurately, with standard deviation values of 1.905 cycles on average, 1.36 for Prunus persica, and 2.45 for other Prunus species tested. The results of this study support the need to use more than one housekeeping gene as an internal control to avoid possible errors caused by unstable internal control gene mRNA expression when quantifying the extent of PDV infection.

摘要

描述了一种使用 SYBR Green 为基础的工具的定量 PCR、实时 RT-PCR 和一步实时 RT-PCR，可用于可靠检测和相对定量李属矮缩病毒（PDV）在核果中的存在。该检测方法在来自不同宿主和地区的 55 个样本中进行了可靠性测试。还将该检测方法的灵敏度与其他使用不同引物的检测方法进行了比较。两种植物表达基因，肌动蛋白和 18S rRNA，被用作核果树木中 PDV 准确定量的管家基因。肌动蛋白基因和 18S 核糖体 RNA 基因的表达彼此准确对应，平均标准偏差值为 1.905 个循环，桃的标准偏差值为 1.36，其他测试的李属物种的标准偏差值为 2.45。本研究的结果支持需要使用多个管家基因作为内参，以避免在定量 PDV 感染程度时，由于不稳定的内参基因 mRNA 表达可能导致的错误。