甘蔗宿根矮化病菌实时荧光定量PCR检测方法的建立

 甘蔗宿根矮化病是由Leifsonia xyli subsp. xyli (Lxx)引起的一种世界性甘蔗细菌病害。根据Lxx的Pat1基因保守序列，设计并合成了一对特异性引物Pat1F (5′-GGTTCCATTGCTTACCGATT-3′)/Pat1R(5′-CAAGTTTCGACAGGAACAGC-3′)，和一条TaqMan探针(FAM-5′-CCACGGCTACGTCAATTCGGG-3′-TAMRA)，建立了一种特异性强、灵敏度高的甘蔗宿根矮化病菌实时荧光定量PCR检测方法。结果表明，本研究建立的实时荧光定量PCR方法，对Lxx的检测最低下限为10² copies·μL^-1。应用实时荧光定量PCR与常规PCR方法对14个甘蔗品种进行Lxx检测，阳性检出率分别为86%和43%，表明实时荧光定量PCR比常规PCR检测方法具有更高的灵敏度。研究结果为甘蔗宿根矮化病的诊断、田间发生动态监测、脱毒健康种苗检测及品种/材料交换检疫检测提供了新技术支撑。

Development of a real-time fluorescence quantitative PCR assay for detection of Lei-fsonia xyli subsp. xyli in sugarcane

Leifsonia xyli subsp. xyli (Lxx) is the pathogen causing ratoon stunting disease of sugarcane and resulting in a considerable reduction in cane production. In this study, a TaqMan probe and a pair of primers were designed according to the Pat1 gene sequence of Lxx for detection of Lxx with real-time fluorescent PCR (RT-qPCR) technique. The results showed that only Lxx produced typical amplification curve and the minimum detection limit of RT-qPCR was 10² copies·μL^-1. Fourteen sugarcane varieties from National Regional Test were then used for Lxx detection assay by RT-qPCR and conventional PCR, and the detection level in these two methods were 86% and 43%, respectively, indicating the higher sensitivity of RT-qPCR in detection of Lxx. The study provides rapid, specific and sensitive technical support for diagnosis and monitoring of RSD, and seedling quarantine.