As the genetically modified organisms (GMOs) labeling policies are issued in many countries, qualitative and quantitative polymerase chain reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. Qualitative PCR and TaqMan real-time quantitative PCR methods to detect and identify three varieties of insect resistant cotton, i.e., Mon531 cotton (Monsanto Co.) and GK19 and SGK321 cottons (Chinese Academy of Agricultural Sciences), which were approved for commercialization in China, were developed in this paper. Primer pairs specific to inserted DNAs, such as Cowpea trypsin inhibitor (CpTI) gene of SGK321 cotton and the specific junction DNA sequences containing partial Cry1A(c)gene and NOSterminator of Mon531, GK19, and SGK321 cotton varieties were designed to conduct the identified PCR assays. In conventional specific identified PCR assays, the limit of detection (LOD) was 0.05% for Mon531, GK19, or SGK321 in 100 ng of cotton genomic DNA for one reaction. Also, the multiplex PCR method for screening the three GM cottons was also established, which could save time and cost in practical detection. Furthermore, a real-time quantitative PCR assay based on TaqMan chemistry for detection of insect resistant gene, Cry1A (c), was developed. This assay also featured the use of a standard plasmid as a reference molecule, which contained both a specific region of the transgene Cry1A (c) and an endogenous stearoyl-acyl carrier protein desaturase (Sad1) gene of the cotton. In quantitative PCR assay, the quantification range was from 0.01 to 100% in 100ng of the genome DNA template, and in the detection of 1.0, 3.0, and 5.0% levels of three insect resistant cotton lines, respectively, all of the relative standard deviations (RSDs) were less than 8.2% except for the GM cotton samples with 1.0% Mon531 or GK19, which meant that our real-time PCR assays involving the use of reference molecule were reliable and practical for GM insect resistant cottons quantification. All of these results indicated that our established conventional and TaqMan real-time PCR assays were applicable to detect the three insect resistant cottons qualitatively and quantitatively.

A novel real-time quantitative polymerase chain reaction (PCR) method using an attached universal template (UT) probe is described. The UT is an approximately 20 base attachment to the 5

Genetically modified (GM) tomatoes have approved for commercialization in many countries since the first GM tomato FLAVER AVAR was permitted for planting in 1994. In 1994. In China, GM tomato Huafan No1 with a character of long shelf-life was the first GM plant which was approved forcommercialization in 1996. To meet the require ment of the GM tomatoes labeling policy that has been universal elements transformed into tomato, such as cauliflower mosaic virus 35S (CaMV35s) promoter and the nopaline synthase ●(NOS) terminator of Agrobacterium tumefaciens, and the specifically inserted heterologous DNA sequence between CaMV35s promoter and anti-sense ethylene-forming enzyme ●(EFE) gene were set up. To make the detection methods normative, a novel single copy tomato gene LAT52 was also used as an endogenous reference gene in the PCR detection systems. The limit of detection of screening and construct specific detection methods for Huafan No1 was 68 haploid genome copies in conventionl PCR detection, and three copies in TaqMan real-time PCR detection. The limit of quantitation of screening quantitative PCR assays for Huafan No1 was three copies and was 25 copies for construct-specific quantitative PCR. Two samples with know Huafan No1 was three copies and was 25 copies for construct-specific quantitative PCR. Two samples with known Huafan No1 tomato content were detected using the established conventional and real-time PCR systems, and these results also indicted that the established Huafan No1 screening and construct-specific PCR detection systems were reliable, sensitive and accurate.

Transgenic tobacco plants stably expressing recombinant FaeG, which is the major subunit and adhesin of K88ad fimbriae, were obtained. Analysis of sera from immunized mice indicates that in mice, the immunogenicity induced by plant-derived FaeG protein is comparable to that generated with traditional approaches.

With the development of transgenic crops, many countries have issued regulations to label the genetically modified organisms (GMOs) and their derived products. Polymerase Chain Reaction (PCR) methods are thought to be reliable and useful techniques for qualitative and quantitative detection of GMOs. These methods generally need to amplify the transgene and compare the amplified result with that of the corresponding reference gene to obtain reliable results. In this article, we reported the development of specific primers and probe for the rice (Oryza sativa) sucrose phosphate synthase (SPS) gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 13 different rice varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other species, such as wheat, maize, barley, tobacco, soybean, rapeseed, tomato, sunflower, carrot, pepper, eggplant, lupine, mung bean, plum, and Arabidopsis thaliana, were used as templates, which demonstrated that this system was specific for rice. In addition, the results of the Southern blot analysis confirmed that the SPS gene was a single copy in the tested rice varieties. In qualitative and quantitative PCR analyses, the detection sensitivities were 0.05 and 0.005ng of rice genomic DNA, respectively. To test the practical use of this SPSgene as an endogenous reference gene, we have also quantified the,-glucuronidase (GUS) gene in transgenic rice using this reference gene. These results indicated that the SPSgene was species specific, had one copy number, and had a low heterogeneity among the tested cultivars. Therefore, this gene could be used as an endogenous reference gene of rice and the optimized PCR systems could be used for practical qualitative and quantitative detection of transgenic rice.