Junyi Xu

Loop-mediated isothermal amplification (LAMP) method for detection of genetically modified maize T25.

The loop-mediated isothermal amplification (LAMP) assay indicates a potential and valuable means for genetically modified organism (GMO) detection especially for its rapidity, simplicity, and low cost. We developed and evaluated the specificity and sensitivity of the LAMP method for rapid detection of the genetically modified (GM) maize T25. A set of six specific primers was successfully designed to recognize six distinct sequences on the target gene, including a pair of inner primers, a pair of outer primers, and a pair of loop primers. The optimum reaction temperature and time were verified to be 65°C and 45 min, respectively. The detection limit of this LAMP assay was 5 g kg−1 GMO component. Comparative experiments showed that the LAMP assay was a simple, rapid, accurate, and specific method for detecting the GM maize T25.

Flanking sequence determination and event-specific detection of genetically modified wheat B73-6-1

In order to establish a specific identification method for genetically modified (GM) wheat, exogenous insert DNA and flanking sequence between exogenous fragment and recombinant chromosome of GM wheat B73-6-1 were successfully acquired by means of conventional polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR strategies. Newly acquired exogenous fragment covered the full-length sequence of transformed genes such as transformed plasmid and corresponding functional genes including marker uidA, herbicide-resistant bar, ubiquitin promoter, and high-molecular-weight gluten subunit. The flanking sequence between insert DNA revealed high similarity with Triticum turgidum A gene (GenBank: AY494981.1). A specific PCR detection method for GM wheat B73-6-1 was established on the basis of primers designed according to the flanking sequence. This specific PCR method was validated by GM wheat, GM corn, GM soybean, GM rice, and non-GM wheat. The specifically amplified target band was observed only in GM wheat B73-6-1. This method is of high specificity, high reproducibility, rapid identification, and excellent accuracy for the identification of GM wheat B73-6-1.

Detection of shrimp-derived components in food by real-time fluorescent PCR.

Crustaceans such as shrimp and crabs and their products are important allergens in food, and allergic reactions due to the consumption of shrimp and crabs are frequently reported. However, the chemical properties of shrimp-derived allergens, except for Pen a I, are still unclear. Therefore, it is important to establish a more sensitive and specific method for detecting the composition of foods containing shrimp. In the present study, we developed a real-time fluorescent PCR to identify the specific shrimp-derived components in food. The primers and TaqMan probes for real-time fluorescent PCR were designed based on 16S rRNA genes through comparing a large number of nucleic acid sequences from different species of shrimp that have been published by the National Center for Biotechnology Information. In total, 56 kinds of samples, including different kinds of shrimp, crab, fish, shellfish, and octopus, were subjected to detection by real-time PCR. The results indicated that real-time fluorescent PCR could successfully identify the shrimp-derived components. In order to explore the effect of food processing on detection sensitivity, fish powder containing shrimp powder was treated by heating at 133°C for 30 min. The limit of detection of shrimp-derived components in fish powder was 0.05% (wt/wt).

Flanking sequence determination and specific PCR identification of transgenic wheat B102-1-2.

The exogenous fragment sequence and flanking sequence between the exogenous fragment and recombinant chromosome of transgenic wheat B102-1-2 were successfully acquired using genome walking technology. The newly acquired exogenous fragment encoded the full-length sequence of transformed genes with transformed plasmid and corresponding functional genes including ubi, vector pBANF-bar, vector pUbiGUSPlus, vector HSP, reporter vector pUbiGUSPlus, promoter ubiquitin, and coli DH1. A specific polymerase chain reaction (PCR) identification method for transgenic wheat B102-1-2 was established on the basis of designed primers according to flanking sequence. This established specific PCR strategy was validated by using transgenic wheat, transgenic corn, transgenic soybean, transgenic rice, and non-transgenic wheat. A specifically amplified target band was observed only in transgenic wheat B102-1-2. Therefore, this method is characterized by high specificity, high reproducibility, rapid identification, and excellent accuracy for the identification of transgenic wheat B102-1-2.