葡萄灰霉病菌(Botrytis cinerea)的快速分子检测

Rapid Molecular Detection of Botrytis cinerea by PCR

为建立快速检测葡萄灰霉病菌的方法,根据ITS序列的差异,设计了1对葡萄灰霉病菌PCR检测的特异性引物TBCF/TBCR,并利用该引物通过常规和巢式PCR对葡萄灰霉病菌进行检测。结果显示,在优化的反应体系与扩增条件下,只有以葡萄灰霉病菌基因组DNA为模板的扩增体系中具有1条386 bp的条带,而其它病菌不产生扩增反应;其常规PCR对葡萄灰霉病菌基因组DNA的检测灵敏度为100 pg/25μL,而利用引物ITS1/ITS4和TBCF/TBCR通过巢式PCR扩增,其灵敏度可达10 fg/25μL。研究表明建立的PCR检测方法具有特异性强、灵敏度高、检测准确而操作简单快速等特点,可用于带菌土壤及发病组织中葡萄灰霉病菌的检测。

In order to develop a technique for the rapid detection of Botrytis cinerea,we designed a pair of specific primers（TBCF/TBCR） for B. cinerea according to the sequence difference of internal transcribed spacer region（ITS） of the ribosomal gene,and used these primers to detect B. cinerea through conventional PCR and nest-PCR. The results indicated that：under optimized reaction system and amplification conditions,only a single band（386 bp） of PCR product was amplified from B. cinerea through the primers TBCF/TBCR,and the other tested pathogens were negative. The detection sensitivity of conventional PCR for the genomic DNA of B. cinerea was 100 pg/25 μL,while that of nest-PCR with the primers ITS1/ITS4 and TBCF/TBCR was 10 fg/25 μL. This constructed PCR detection method is characterized by strong specificity,high sensitivity,high accuracy,and easy and rapid operation,so it can be used to detect B. cinerea in soil and diseased grape tissues.