小麦全蚀病菌重组酶聚合酶检测方法的建立及应用

Development and evaluation of recombinase polymerase amplification for the detection of Gaeumannomyces graminis var.tritici

由禾顶囊壳小麦变种Gaeumannomyces graminis var.tritici引起的小麦全蚀病是危害极大的小麦土传真菌病害。本研究以小麦全蚀病菌β-tubulin为靶标基因设计重组酶聚合酶扩增技术(recombinase polymerase amplification,RPA)引物Gg-RPA-F/Gg-RPA-R和RPA探针Gg-LF-Probe,结合侧流层析试纸条,建立了小麦全蚀病菌RPA快速检测方法,该方法在38℃恒温条件下30 min内完成可视化检测,摆脱PCR仪等仪器设备的限制。小麦全蚀病菌RPA快速检测方法具有较高的检测特异性,其检测极限为10 pg/μL,与普通PCR一致。此外,小麦全蚀病菌RPA快速检测方法可从土壤中快速检测到小麦全蚀病菌,具有较强的实用性。因此,本研究建立的小麦全蚀病菌RPA快速检测方法具备简便高效、实用性强的特点,为小麦全蚀病菌的快速检测和病害早期诊断提供新技术。

Gaeumannomyces graminis var.tritici,is a soil-borne phytopathogenic fungus that causes wheat take-all,resulting in serious economic losses.In this study,the recombinase polymerase amplification(RPA)combined with lateral-flow dipstick technology was developed for the rapid and sensitivity detection of G.graminis var.tritici.The RPA assay could be completed at isothermal temperature of 38℃within 30 min without PCR thermal cycles.The RPA primers(Gg-RPA-F/Gg-RPA-R)and probe(Gg-LF-Probe)designed based on theβ-tubulin gene,which showed high specificity to G.graminis var.tritici.The detection limit of RPA assay was 10 pg/μL fungal genomic DNA,demonstrating an equal sensitivity to that of conventional PCR.In addition,the RPA assay could detect G.graminis var.tritici from field soil samples.The simplicity,rapidly and practicality all indicated that RPA assay will be a promising molecular diagnosis for the accurate and rapid detection of G.graminis var.tritici.