小麦根腐病菌索氏平脐蠕孢SYBR Green I实时荧光定量PCR检测技术研究

Quantitative detection of Bipolaris sorokiniana in winter wheat based on SYBR Green I realtime PCR

由索氏平脐蠕孢Bipolaris sorokiniana引起的小麦根腐病,常和其他土传真菌病害混合发生,传统的症状鉴别方法很难区分,导致病害防控难度增加。为建立病菌实时荧光定量检测体系,根据ITS序列设计引物,筛选出1对特异性引物BS‐F/R,扩增片段大小为280bp。以菌丝DNA为标准品构建实时荧光定量标准曲线,并对其灵敏度、特异性、可重复性进行评价。结果表明,建立的实时荧光定量PCR检测方法速度快,灵敏度高,特异性强,重复性好。构建的荧光定量PCR标准曲线循环阈值与模板浓度呈良好的线性关系,溶解曲线的吸收峰单一,扩增效率良好。利用该定量检测体系,可以检测出田间小麦样品中52.8fg/μL的病菌DNA。

The common root rot of wheat, caused by BipoJoris sorokiniono, always occurs together with other soil-borne diseases showing similar symptoms. Traditional methods based on symptoms are unsuitable for Bipoloris sorokiniana identification, leading to the increased difficulty for disease control. In order to timely detect the pathogen, real-time PCR was carried out with specific primers which were designed according to rDNA ITS sequence. The pair of primers, BS-F/R, amplified the fragment of 280bp in length were screened out. After optimization of real-time PCR programme, a standard curve was established using pure mycelium DNA of the pathogen and meanwhile the sensitivity, specificity and repeatability of the reaction were analyzed. The cycle threshold of standard curve has a good linear relationship with template concentration of pathogenic DNA, and the PCR amplification generated a single absorption peak in melting curves. The DNA of the pathogen was detectable at the concentration of 52.8fg/μL in infected wheat.