Tian Yanli, Zhao Yuqiang, Xu Rui, Liu Fengquan, Hu Baishi, Walcott R R
Phytopathology. 2014 Oct;104(10):1130-7. doi: 10.1094/PHYTO-10-13-0274-R.
Based on 16S-23S internal transcribed spacer ribosomal DNA sequence data, two padlock probes (PLPs), P-Xoo and P-Xoc, were designed and tested to detect Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola, respectively. These PLPs were combined with dot-blot hybridization to detect X. oryzae pv. oryzae and X. oryzae pv. oryzicola individually in rice seed. Using this technique, a detection sensitivity of 1 pg of X. oryzae pv. oryzae genomic DNA was observed. The technique also facilitated the detection of X. oryzae pv. oryzae in rice seedlots with 2% artificially infested seed. With regards to X. oryzae pv. oryzicola a detection threshold of 1 pg genomic DNA was observed and the pathogen was successful detected in rice seedlots with 0.2% artificially infested seed. The PLP assays detected X. oryzae pv. oryzae and X. oryzae pv. oryzicola in 39.3% (13 of 33) and 21.3% (10 of 47) of naturally infested commercial rice seedlots, respectively. In contrast, conventional polymerase chain reaction using OSF1/OSR1 and XoocF/XoocR primers sets detected X. oryzae pv. oryzae and X. oryzae pv. oryzicola in 9.1% (3 of 33) and 8.5% (4 of 47) of the same rice seedlots, respectively. We also detected both pathogens simultaneously in two seedlots, which successfully proved that PLPs (P-Xoo and P-Xoc) combined with reverse dotblot hybridization can be used to simultaneously detect multiple pathogens in naturally infested commercial rice seedlots. This approach has the potential to be an important tool for detecting multiple pathogens in seed and thereby preventing the spread of important pathogens.
基于16S - 23S内转录间隔区核糖体DNA序列数据，设计并测试了两种锁式探针（PLP），即P - Xoo和P - Xoc，分别用于检测水稻白叶枯病菌和水稻细菌性条斑病菌。这些锁式探针与斑点杂交相结合，以单独检测水稻种子中的水稻白叶枯病菌和水稻细菌性条斑病菌。使用该技术，观察到对1 pg水稻白叶枯病菌基因组DNA的检测灵敏度。该技术还便于检测含有2%人工接种种子的水稻种子批中的水稻白叶枯病菌。对于水稻细菌性条斑病菌，观察到1 pg基因组DNA的检测阈值，并且在含有0.2%人工接种种子的水稻种子批中成功检测到该病原菌。锁式探针检测法分别在39.3%（33个中有13个）和21.3%（47个中有10个）的自然感染商业水稻种子批中检测到水稻白叶枯病菌和水稻细菌性条斑病菌。相比之下，使用OSF1/OSR1和XoocF/XoocR引物组的传统聚合酶链反应分别在相同水稻种子批的9.1%（33个中有3个）和8.5%（47个中有4个）中检测到水稻白叶枯病菌和水稻细菌性条斑病菌。我们还在两个种子批中同时检测到了这两种病原菌，这成功证明了锁式探针（P - Xoo和P - Xoc）与反向斑点杂交相结合可用于同时检测自然感染商业水稻种子批中的多种病原菌。这种方法有可能成为检测种子中多种病原菌从而防止重要病原菌传播的重要工具。
