
Strains of
Whole genome shotgun sequencing of
The genomic DNA was extracted from the bacterial cells using the GeneAll ExgeneTM Cell SV kit (GeneAll Biotechnology, Seoul, Korea) according to the manufacturer’s protocol. DNA concentrations were determined with a QubitTM fluorometer (Invitrogen®, Carlsbad, CA, USA).
The PCR assays used 1 μl of template DNA in a 25 μl reaction mixture containing 2 mM of Tris-HCl (pH 8.0), 10 mM of KCl, 10 uM of EDTA, 100 uM of DTT, 0.05% Tween 20, 0.05% Nonidet P-40, 5% glycerol, 2.5 mM of dNTP, 5 units of Ex Taq DNA (TaKaRa Bio Inc., Shiga, Japan), and 10 pM of each primer. The reactions were performed in a T Gradient Thermal Cycler (Biometra, Göttingen, Germany) programmed for one cycle of 10 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C, with a final extension step for 7 min at 72°C. The amplicons were analyzed by electrophoresis in a 2% agarose gel in TAE buffer.
The TaqMan PCR assay was carried out using 2 μl of DNA template, 8.5 μl of Premix Ex Taq (TaKaRa Bio Inc., Shiga, Japan), 1 μl of TaqMan probe (10 pmoles/μl), 0.5 μl (10 pmoles/μl) of each nested primer, and 12.5 μl of DEPC-DW (Bioneer, Daejeon, Korea). Real-time PCR amplifications were performed in a Smart CyclerTM System (Cepheid, Sunnyvale, CA, USA) with 40 cycles at 95°C for 30 sec and 60°C for 20 sec after initial incubation for 30 sec at 95°C.
Prior to inoculation, the oat seeds (cultivar Samhan, provided by the National Honam Agricultural Experiment Station in Korea) were surface sterilized by soaking in 70% ethanol for 30 minute and 1.2% sodium hypochlorite solution for 1 hour. The oats seeds were washed 3 times with sterilized water and dried completely in clean bench. Twelve grams of the surface-sterilized oat seeds were artificially inoculated by shaking (120 rpm) in 45 ml of
To obtain specific primers of the
To reduce the rate of false-positives and quantitatively detect
The specificity of the TaqMan PCR was checked with the target and non-target bacteria. In the TaqMan PCR assays, 10-fold diluted DNAs (10 ng to 10 pg) of
The TaqMan PCR assay developed in this study was applied to detect
Estimation of the recovery number of P. coronafaciens from the artificial inoculated seed extract was tried with several different ways, but the accurate enumeration of
Highly specific PCR and TaqMan PCR assays have been developed in this study to detect
To obtain specific primers of the
TaqMan PCR was applied to detect the target pathogen from artificially inoculated oat seeds. TaqMan PCR generated the