茄匍柄霉菌实时荧光定量PCR检测方法的建立及应用

 为了准确检测病残体内茄匍柄霉菌(Stemphylium solani)DNA含量在土壤内的动态变化,本研究根据三磷酸甘油醛脱氢酶(GAPDH)基因序列,设计并筛选特异性引物Stem-g7F/Stem-g7R,能从靶标基因组 DNA 中特异性扩增出大小为 150 bp 的目的片段。建立的Stemphylium solani实时荧光定量PCR(Real-time quantitative PCR, qRT-PCR)检测体系的灵敏度比常规 PCR 高 1 000 倍,且特异性良好。标准曲线循环阈值与模板浓度呈良好的线性关系,相关系数为 0.992。利用所建立的qRT-PCR方法对病残体进行检测发现,病残体DNA初始拷贝数为3.69 × 10⁹ 拷贝数/g,经过温度27℃、80%湿度下处理30 d病残体DNA含量下降至1.21 × 10⁶ 拷贝数/g,而在温度27℃、20%湿度下含量为1.29 × 10¹⁰ 拷贝数/g。因此,建立的S. solani的qRT-PCR检测体系具有特异性强、灵敏度高的特点,可以快速准确地定量检测病残体 S. solani 的含量,为番茄匍柄霉叶斑病的早期预防和流行监测提供有效的技术手段。

DNA detection and quantification of Stemphylium solani in heat-treated infected leaf tissues in vary moisture conditions using real-time qPCR

In order to accurately evaluate the level changes of the Stemphylium solani associated with diseased tomato debris in the soil, a real-time quantitative PCR assay for quantitative detection of Stemphylium DNA in tomato leaf spot was developed. Based on the gene sequence encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in S. solani, a Stem-g7F/Stem-g7R primer pair was designed for which a 150 bp fragment was specifically amplified from the genomic DNA as template. The sensitivity of real-time quantitative PCR established in this study was 10³ times higher than that of conventional PCR. There was a good linear(R²= 0.992)relationship between the threshold cycle and the template concentration. The results showed that the initial copy numbers of DNA in crop residues was 3.69 × 10⁹copies/g. After treatment at 27℃ with humidity 80% for 30 days, the limit of DNA detection from the leaf tissues was decreased to 1.21 × 10⁶ copies/g, in contrast that of 1.29 × 10¹⁰copies/g at the relative humidity of 20%. Therefore, qRT-PCR assay is a highly rapid and reliable method to quantify S. solani in the artificial infected tomato debris. Application of the assay for the fungal detection and quantification may potentially improve the prevention for tomato leaf spot disease at the earlier stage and management on the dynamic of disease spread.