论文部分内容阅读
为建立猕猴桃果实表皮溃疡病病原菌(Psa)的快速检测方法,以陕西周至县贮藏期的海沃德猕猴桃为材料,用King’s B液体培养基培养获得果实表面病原菌,采用细菌DNA提取试剂盒及热裂解法提取总DNA;根据陕西猕猴桃Psa的hrp W保守区设计2对特异引物进行巢式PCR扩增,并对扩增产物进行测序。结果表明,从4个猕猴桃贮藏库抽取40个样品,其中8个样品检测出阳性扩增;对特异扩增得到的550bp的hrp W序列进行BLAST对比分析,发现该序列与丁香假单胞杆菌猕猴桃致病性变种(Pseudomonas syringae pv.actinidiae)的hrp W序列同源性为99%。此外,本试验建立的巢式PCR检测方法对Psa的检出限为103cfu·m L~(-1),检测方法特异性强,灵敏度高,能减少假阳性的出现,保证了快速检测结果的可靠性,为控制猕猴桃果实传播Psa提供了理论依据。
In order to establish a rapid method for the detection of kiwifruit fruit skin ulcer disease pathogen (Psa), kiwifruit kiwifruit stored in Zhouzhi County, Shaanxi Province was used as material to cultivate fruit surface pathogens in King’s B liquid medium. Bacterial DNA extraction kit and heat According to the hrp W conserved region of Actinidia chinensis Psa, two pairs of specific primers were designed for nested PCR amplification, and the amplified products were sequenced. The results showed that 40 samples were extracted from 4 kiwifruit banks, of which 8 samples were detected positive amplification; BLAST analysis of the 550 bp hrp W sequence obtained by specific amplification showed that this sequence was closely related to the Pseudomonas syringae kiwifruit The hrp W sequence homology of the pathogenic Pseudomonas syringae pv. Actinidiae was 99%. In addition, the detection limit of Psa established by this method was 103 cfu · m L -1. The detection method is specific and sensitive, and can reduce the false positives and ensure the rapid detection of Reliability, provide a theoretical basis for the control of Kiwifruit fruit spread Psa.