Key Laboratory of Biorheological Science and Technology, College of Biological Engineering, Chongqing University, Chongqing 400044, China.
Microbiol Res. 2010 Jul 20;165(5):411-7. doi: 10.1016/j.micres.2009.09.001. Epub 2009 Oct 17.
Thielaviopsis basicola is a soil-borne fungus with a wide host range and a cosmopolitan distribution. It causes disease on many agricultural crops and in China it is the causal agent of black root rot on tobacco plant. Early diagnosis and detection of the pathogen in soil are critical to control this disease in field. The objective of this study was to develop sensitive and effective methods suitable for large-scale detection and quantification of T. basicola. Based on the nucleotide sequences of the internal transcribed spacer (ITS) regions of rDNA genes of Thielaviopsis spp, primers and TaqMan probe were designed specifically to amplify DNA from T. basicola and real-time, quantitative PCR (qPCR) assays were developed for rapid, specific and sensitive detection and quantification of T. basicola. It was sensitive with the detection limit of 100 fg microl(-1) genomic DNA of T. basicola in qPCR assays. By combining the qPCR assays with the efficient protocol to extract DNA from soil, it was possible to achieve real-time detection of T. basicola in soil in 4-5 h and the detection limit of 3 conidia per reaction in qPCR was recorded. The assays were applied to survey soils from tobacco fields in China for the presence of T. basicola and the analyses of naturally infested soil showed the reliability of the qPCR assays.
基腐隔孢腔菌是一种土壤传播真菌，具有广泛的宿主范围和世界性分布。它可引起许多农作物病害，在中国是烟草黑胫病的病原体。早期诊断和检测土壤中的病原体对于控制田间病害至关重要。本研究旨在开发敏感且有效的方法，适用于大规模检测和定量基腐隔孢腔菌。基于 Thielaviopsis spp 的 rDNA 基因内转录间隔区（ITS）区域的核苷酸序列，专门设计了引物和 TaqMan 探针，用于扩增 T. basicola 的 DNA，并开发了实时定量 PCR（qPCR）检测方法，用于快速、特异性和敏感地检测和定量 T. basicola。该方法在 qPCR 检测中具有 100 fg microl(-1)基因组 DNA 的检测限。通过将 qPCR 检测与从土壤中提取 DNA 的高效方案相结合，有可能在 4-5 小时内实时检测土壤中的 T. basicola，记录到 qPCR 中每个反应的检测限为 3 个分生孢子。该检测方法应用于中国烟草田土壤中 T. basicola 的存在调查，自然感染土壤的分析表明 qPCR 检测方法的可靠性。
