Environmental Health Program (Biodiversity), Agriculture and Agri-Food Canada, Ottawa, ON, Canada.
Lett Appl Microbiol. 2010 Feb;50(2):216-22. doi: 10.1111/j.1472-765X.2009.02780.x. Epub 2009 Nov 24.
Development of a 'miniprimer' PCR assay for genotyping Pantoea stewartii subsp. stewartii, the causal agent of the Stewart's bacterial wilt on maize.
Four 10-nucleotide (10-nt) 'miniprimer' sets were designed and evaluated in the presence of Titanium Taq DNA polymerase. Under optimal reaction conditions, the miniprimer pair Uni-BacF-10/Uni-BacR-10 reproducibly generated identical banding patterns among 10 strains of P. stewartii subsp. stewartii, different patterns from strains of P. stewartii subsp. indologenes, other Panteoa species, Clavibacter michiganensis, Pectobacterium spp., Pseudomonas spp. and other bacterial species. The amplicons of Pantoea stewartii subsp. stewartii were cloned and sequenced to identify genes or DNA fragments that are targeted by the miniprimer PCR assay. Of the 14 'clone types' identified, sequences of a 1.23-kb fragment had a 99.8% similarity to part of the Pantoea stewartii zeaxanthin diglucoside biosynthetic operon (AY166713). Other dominant cloned fragments included a 411-bp amplicon that exhibited 99.8% similarity to the psaU gene (syn:ysaU; GQ249669), a type III protein-secretion system complex of P. stewartii subsp. stewartii strain DC283, and a 548-bp fragment showed 63% homology to the Asp/Glu racemase encoding gene in Erwinia tasmaniensis strain ET1/99.
The miniprimer PCR assay reported here is highly discriminatory and reproducible in genotyping Pantoea stewartii subsp. stewartii.
This miniprimer PCR assay could be a new reliable and rapid tool for fingerprinting the Stewart's wilt pathogen of maize.
开发一种用于 Pantoea stewartii subsp. stewartii 基因分型的“微型引物”PCR 检测方法，Pantoea stewartii subsp. stewartii 是引起玉米细菌性枯萎病的病原体。
设计了四组 10 个核苷酸（10-nt）的“微型引物”组，并在 Titanium Taq DNA 聚合酶存在的情况下进行了评估。在最佳反应条件下，微型引物对 Uni-BacF-10/Uni-BacR-10 可在 10 株 P. stewartii subsp. stewartii 菌株中产生相同的条带模式，与 P. stewartii subsp. indologenes 菌株、其他 Pantoea 物种、Clavibacter michiganensis、Pectobacterium spp.、Pseudomonas spp. 和其他细菌物种的模式不同。克隆并测序 Pantoea stewartii subsp. stewartii 的扩增子，以鉴定微型引物 PCR 检测方法所针对的基因或 DNA 片段。在所鉴定的 14 种“克隆类型”中，长度为 1.23kb 的片段序列与 Pantoea stewartii 玉米黄质双葡萄糖苷生物合成操纵子（AY166713）的一部分具有 99.8%的相似性。其他主要的克隆片段包括 411bp 的扩增子，其与 P. stewartii subsp. stewartii 菌株 DC283 的 psaU 基因（同义：ysaU；GQ249669）表现出 99.8%的相似性，以及一种 III 型蛋白分泌系统复合物，和一个 548bp 的片段与 Erwinia tasmaniensis 菌株 ET1/99 的 Asp/Glu 消旋酶编码基因具有 63%的同源性。
本研究报道的微型引物 PCR 检测方法在 Pantoea stewartii subsp. stewartii 基因分型中具有高度的区分性和重现性。
这种微型引物 PCR 检测方法可能成为一种用于鉴定玉米细菌性枯萎病病原体的新的可靠、快速工具。
