Biotechnology has permitted the modification of agricultural materials in a very precise way to improve productivity and yields. Polymerase chain reaction (PCR)-based methods have been the first choice of most analytical laboratories for routine use in the detection of genetically modified organisms (GMO) and their derived products. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison with an amplified reference gene. This paper describes the specific primers and probe for the cotton stearoyl-ACP desaturase (sad1) gene, and PCR cycling conditions suitable for the use of this sequence, which acts as an endogenous reference gene in both qualitative and quantitative PCR assays. The two methods were tested with 18 cotton varieties and identical amplification products were obtained with all of them. No amplification products were detected when DNA samples from other species, including soybean, rapeseed, tobacco, maize, tomato, potato, cucumber, pea, red pepper, sunflower, sesame, rice, peach, banana, apple, pumpkin, barley and carrot, were used as templates, which demonstrates that this system is specific for cotton. In real-time quantitative PCR analysis, the detection limit was as low as 6 pg of DNA, which indicates that this method is suitable for application to processed food samples that contain very low copies of target DNA. Southern blot analysis confirmed that the sad1 gene was a single copy in the tested cotton varieties.  2006 Society of Chemical Industry

The application of Grapefruit Seed Extract (GSE) as a sanitizer for reducing the populations of human bacterial pathogens on whole and fresh-cut green vegetables was investigated. Cucumber and lettuce were selected as model green vegetables, and six bacteria strains, including three strains of Salmonella spp. and three strains of Listeria monocytogenes, were selected in our study in order to determine the antibacterial effects of sanitizers. The survival and growth of total aerobes, Salmonella spp., and L. monocytogenes on whole and fresh-cut cucumber and lettuce during storage (10 and 4℃) were analyzed by using a classical microbiological enumeration. The antibacterial effects of GSE alone and GSE in combination with nisin and citric acid (Mixture agent II) were significant (P < 0.05). Treatments with sodium lactate and potassium sorbate (Mixture agent I) were also tested as chemically synthesized agents. Sensory quality was evaluated, and there was no significant difference between GSE and Mixture agent II treatments during storage in terms of organoleptic and visual properties. Our results suggest that GSE could inhibit bacteria significantly (P < 0.05) and prolong the preservation time; GSE might be applied as an effective and safe preservative for ready-to-eat cucumber and lettuce.

We have developed a new immunoassay method to detect genetically modified (GM) maize and rape containing phosphinothricin-N-acetyltransferase (PAT). PAT encoded by Bialaphos resistance gene (bar) was highly expressed in soluble form in Escherichia coli BL21(DE3) and purified to homogeneity by Ni2+ affinity chromatography. A simple and efficient extraction and purification procedure of PAT from GM maize and rape was developed by means of the immunoaffinity column (IAC) as a cleanup tool. Purified polyclonal antibodies against PAT was produced and coupled covalently to CNBr-activated Sepharose 4B. Both the binding conditions and elution protocols were optimized. The IAC was successfully employed to isolate and purify the PAT from the various tissues of GM maize (Bt11 and Bt176) and rapes (MS1/RF1 and MS8/RF3). Enzyme linked immunosorbent assay (ELISA) procedures were established further on to measure the PAT protein. GM maize cannot be differentiated from non-GM maize by ELISA. But IAC-ELISA allowed 0.5% GMOs to be detected in MS1/RF1 and MS8/RF3 and 10% GMOs to be detected in Bt11 and Bt176, which makes this method an acceptable method to access PAT protein in GM rapes and maize.

The present study investigated the DNA density of the embryo, cotyledon, and seed coat of each soybean from 15 soybean cultivars, and evaluated the impacts of variations of tissue DNA density and nuclear DNA content in soybean lines on GMO quantification. The results have shown that DNA densities and DNA quantity ratios among the various tissues of soybean are significantly different from each other and have insignificant influence on the transgenic copy number and therefore on GMO quantification. The nature of the CRM and the transgenic material present in the analyzed sample has prominent impact on transgenic genome copies, thus on GMO quantification. The DNA densities of the various soybean-derived products are also distinctly different from that of the whole soybean, but which does not result in the similar differences on the GMO ratio among them. Nuclear DNA content of soybean is different from cultivar to cultivar. Results shows that variation of nuclear DNA content in soybean lines has a great impact on the accurate determination of GMO. In some extreme situations, the deviation amplitude can reach 26%, which is intolerable for accurate determination. _ 2006 Published by Elsevier Ltd.

With the development of genetically modified organisms, labeling regulations have been introduced, which require appropriate detection methods. Event-specific qualitative and quantitative polymerase chain reaction (PCR) detection methods have become the internationally agreed state-of-art. This paper describes an event-specific PCR method for qualitative and quantitative of Roundup Ready canola event GT73. The 3￠-integration junction was characterized by two methods: inverse-PCR and thermal asymmetric interlaced-PCR. In the conventional qualitative PCR assay, the event-specific primers designed were confirmed to be specific and the limit of detection (LOD) was 0.05% (approximates to ten haploid genome copies). In the quantitative TaqMan real-time PCR assay, the LOD and the limit of quantification were five and ten haploid genome copies, respectively. In addition, for further quantitative detection, a reference molecule which contained the canola endogenous gene and event-specific sequence was constructed and standard curves were set up. The goodness of the linearity and high efficiency of the PCR reaction indicated the usability of the plasmid and the established PCR system. Moreover, mixed samples with different GT73 content (6, 3, 1 and 0.5%) were quantified using the established real-time PCR system to evaluate the trueness and precision of the system. The trueness expressed as bias varied from 2.00 to 18.00%. The precision expressed as variation coefficient were different from 6.40 to 32.95%. From above results, we believed that the established eventspecific qualitative and quantitative PCR systems for GT73 in this study were acceptable and suitable for genetic modified canola detection.