Four chrysanthemum cultivars were generated through (carbon) ion-beam irradiation of the original ‘Jimba’ ( Chrysanthemum morifolium Ramat.). The new cultivars had acquired a number of superior cultivation traits, while remaining identical to the commercially available ‘Jimba’ in appearance. In this study, polymerase chain reaction (PCR) assays were used to detect the mutated region of each strain, thereby allowing clear identification at the molecular level. PCR assays were performed with 446 primer sets, including random amplified polymorphic DNA (RAPD) primer sets (10-mer RAPD), arbitrarily primed (AP)-PCR primers based on retrotransposon-like sequences and modified RAPD primers (15-mer RAPD). 15-mer RAPD primers generated a 1.49-fold increased band number at high annealing temperatures compared with the original 10-mer RAPD primers and could thus be effective for detection of polymorphic patterns. Our results provide information on the mutated regions of these ion-beam-irradiated chrysanthemum cultivars. Thus, specific DNA markers could be used to improve identification of new cultivars of chrysanthemum as well as other clonal cultivars of horticultural and agricultural crops.
Listeriosis outbreaks caused by contaminated leafy greens leaf have increased in recent years, which can pose a serious threat to public health. Thus, a rapid, precise, and effective detection method is urgently needed. We reported a novel sample pre-treatment that utilizes β-cyclodextrin and milk protein-coated activated charcoal to remove PCR inhibitors in leafy green matrices and facilitate the recovery of L. monocytogenes , which can be coupled with PCR subsequently. The primers targeting the iap gene of L. monocytogenes were used for testing the specificity with 141 bacterial strains. Artificially contaminated leafy greens (10 0 –10 5 CFU/25 g) that include spinach, lettuce, Lactuca sativa L., cole, and cabbage were tested to determine the sensitivity of the developed method. Natural leafy greens were tested to evaluate the application of the developed method compared with the conventional culture method. The stability (shelf life) of the reagents used in the developed method was determined using reagents stored at −20 °C for 3, 6, and 12 months to evaluate the sensitivity and application of the method. As a result, the specificity of the developed method was 100% and the detection limit was 10 1 CFU/25 g. The total detection time without pre-enrichment was 4 h. The detection rate of L. monocytogenes (spinach 5%, lettuce 3%, cole 1%, Lactuca sativa L. 0%, and cabbage 0%) and the detection rate of viable bacteria (100%) were consistent in both diagnostic methods (the coincidence rate was 100%). Furthermore, the sensitivity and the application of the developed method were not influenced using the reagents stored for 3, 6, and 12 months. The reagents could be used normally after storing at −20 °C for at least 12 months, which proved that these reagents have good stability under freezing conditions. This method for the detection of L. monocytogenes was rapid, sensitive, and specific, and can potentially be used to detect and monitor the target pathogen in leafy greens and to reduce the potential risk of L. monocytogenes for public health.
危险品化学品经营许可证（带存储）