Tambong J T, Mwange K N, Bergeron M, Ding T, Mandy F, Reid L M, Zhu X
Environmental Health Program (Biodiversity), Agriculture and Agri-Food Canada, Ottawa, ON, Canada.
J Appl Microbiol. 2008 May;104(5):1525-37. doi: 10.1111/j.1365-2672.2007.03674.x. Epub 2007 Dec 20.
The development and evaluation of a sensitive and specific TaqMan real-time polymerase chain reaction (PCR) for the detection and identification of Pantoea stewartii on maize.
A TaqMan-based real-time PCR assay targeting the cpsD gene enabling specific detection of P. stewartii in maize leaves and seeds was developed. Under optimal conditions, the selected primers and probe were specific for the detection of all 14 reference P. stewartii strains by real-time PCR. The 32 non-Panteoa and eight other Pantoea strains tested negative. The TaqMan PCR assay detected 1 pg of purified DNA and 10(4)P. stewartii colony forming units per millilitre (10 cells per reaction) in pure cultures consisting of 92.0% intact (viable) cells. Direct processing of leaf lesions and seeds by the real-time PCR detected 10 and 50 P. stewartii cells per reaction respectively. TaqMan real-time PCR results were validated by dilution plating of macerates and PCR-based subcloning followed by DNA sequencing.
The real-time PCR assay described is a rapid, reliable and more sensitive tool for the detection of P. stewartii.
This real-time PCR assay would avoid false-negative results and reduce the time required for certifying maize seed shipments.
开发并评估一种灵敏且特异的TaqMan实时聚合酶链反应（PCR），用于检测和鉴定玉米上的斯氏泛菌。
开发了一种基于TaqMan的实时PCR检测方法，该方法靶向cpsD基因，能够特异性检测玉米叶片和种子中的斯氏泛菌。在最佳条件下，所选用的引物和探针通过实时PCR对所有14株斯氏泛菌参考菌株进行特异性检测。32株非泛菌属菌株和8株其他泛菌属菌株检测结果为阴性。TaqMan PCR检测方法在由92.0%完整（有活力）细胞组成的纯培养物中能检测到1 pg纯化DNA和每毫升10⁴个斯氏泛菌菌落形成单位（每个反应10个细胞）。通过实时PCR对叶片病斑和种子进行直接检测，每个反应分别检测到10个和50个斯氏泛菌细胞。TaqMan实时PCR结果通过对浸解物进行稀释平板培养以及基于PCR的亚克隆随后进行DNA测序得以验证。
所描述的实时PCR检测方法是一种用于检测斯氏泛菌的快速、可靠且更灵敏的工具。
这种实时PCR检测方法将避免假阴性结果，并减少认证玉米种子运输所需的时间。
