A TaqMan real-time PCR assay targeting the cytochrome o ubiquinol oxidase subunit II gene for detection of several pathovars of Pseudomonas syringae

作者：Xu R; Tambong J T^*

来源：Canadian Journal of Plant Pathology, 2011, 33(3): 318-331.

DOI：10.1080/07060661.2011.600335

• bacterial blight
• bacterial speck
• brown spot
• cyoA
• cytochrome o ubiquinol oxidase
• halo blight
• molecular detection
• polymerase chain reaction
• tomato

The identification and detection of eight pathovars of Pseudomonas syringae, bacterial pathogens of several important agricultural plants, was achieved by TaqMan real-time polymerase chain reaction of a specific DNA fragment of the cytochrome o ubiquinol oxidase gene. Under optimal real-time PCR conditions, the selected primers and probe were specific for the detection of pathovars syringae, tomato, maculicola, tabaci, atropurpurea, phaseolicola, pisi and glycinea. Two pathovars (coriandricola and morsprunorum) tested could be differentiated from the other eight due to a single nucleotide mismatch. Thirty other Pseudomonas strains and 20 non-Pseudomonas strains were negative. The real-time PCR assay detected 100 fg of DNA and 4.5 x 10(3) P. syringae colony forming units per millilitre (four cells per reaction). In growth chamber experiments, tomato plants were inoculated using strain DC3000 and assayed by TaqMan real-time PCR. Serial dilution of leaf extracts spiked with lambda DNA and processed by real-time PCR indicated the presence of inhibitors. A 1:10 dilution of the crude extract reduced threshold cycles to those of milliQ water spiked with the same amount of lambda DNA. The TaqMan real-time assay consistently detected the pathogen in inoculated tomato leaves after a 1:10 dilution of crude extracts. TaqMan real-time results were validated by dilution-plating of leaf extracts and conventional PCR using the same primer set. This assay offers real-time monitoring of the targeted amplicon with high specificity and sensitivity, with no post-amplification analysis needed. This reduces opportunity for contamination of the reaction mixtures with target DNA, making this real-time PCR assay more reliable than conventional PCR. However, for routine diagnosis of the detected pathovars under greenhouse or field conditions, optimization of the assay might be required.

• 出版日期2011