向日葵黑茎病菌RPA/CRISPR-Cas12a快速检测方法的建立

Establishment of RPA/CRISPR-Cas12a rapid detection for Leptosphaeria lindquistii

向日葵黑茎病菌是向日葵上的一种毁灭性真菌,是我国的植物检疫性有害生物。为了准确快速检测向日葵黑茎病菌,本研究根据向日葵黑茎病菌及其近似种的ITS序列差异,设计特异性RPA引物和CRISPR-Cas12a crRNA,建立了RPA等温扩增技术结合CRISPR-Cas12a检测的快速检测方法。通过条件优化,RPA/CRISPR-Cas12a检测体系在37℃恒温条件下,RPA反应30 min,CRISPR/Cas12a反应20 min,即可特异性检测向日葵黑茎病菌。荧光法检测灵敏度与实时荧光PCR的灵敏度相当,最低检测量为0.1 pg,试纸条法检测最低检测量为1 pg。由于试纸条检测结果可用肉眼观察,快速便携,操作简单,更适合用于田间和口岸的向日葵黑茎病菌快速早期检测;而荧光检测灵敏度高,对环境要求高,更适合用于实验室检测。

Leptosphaeria lindquistii is a destructive fungus on sunflowers plants and is a plant quarantine pest in China.In this study,we designed species-specific RPA primers and CRISPR-Cas12 a crRNA based on the ITS sequences of L.lindquistii and its relatives,and developed a combined method of recombinase polymerase amplification(RPA)and CRISPR-Cas12 a system(RPA/CRISPR-Cas12 a),in order to realize the rapid,specific and sensitive detection of L.lindquistii in field or at port.Fluorescent detection and lateral flow assay were developed with fluorescent reporter and lateral flow reporter.Under the optimized conditions,the developed RPA/CRISPR-Cas12 a detection system could specifically detect L.lindquistii at 37℃with 30 min of RPA reaction time and 20 min of CRISPR/Cas12 reaction time.The sensitivity of fluorescent detection is equivalent to that of real-time fluorescent PCR with a low detection limit of 0.1 pg genomic DNA,while the lateral flow assay could detect 1 pg genomic DNA.Since lateral flow assay is visualized,fast,portable,it is feasible to the diagnosis of Leptosphaeria lindquistii at port and in field.The fluorescent detection has higher sensitivity than the lateral flow assay and requires strict environment,so it is more applicable in laboratory.