| In recent years, with the development of bio-technology, the cultivated area of genetically modified crop substantially increase, so people concern its security. Some countries require detecting whether they contain genetically modified ingredients, and some also need to determine the transgenic strains. Genetically modified corn is the world’s second largest growing crops,12kinds of licensing imports of genetically modified maize lines, which are mainly for the processing of raw materials, is the representative of strains of the genetically modified corn. Therefore, the enhance detection of genetically modified crops and their products is very important. For the moment, The tools applied for GMO testing primarily are ptotein based(mainly immunological)assays and DNA based assays (mainly applying the polymerase chain reaction [PCR]technology). In recent years, the gene chip and real-time fluorescence quantitative (the Real-time PCR) detection are widely used. However many genetically modified products are partly or completely degraded after processing and preservation, so the difficulty of detection increased. Therefor an establishment of better, faster, high-throughput method was needed. Liquid chip is a new type of high-throughput detection platform. The benefits of Liquid chip include more accurate, short time-consuming, easy operation, high-throughput, excellent sensitivity and specificity and so on, which is suitable for the quickly detection of a large number of genetically modified crops.This experiment selected four GM maize lines(Bt176, Bt11, NK603and MON810), Based on the sequences of transgenie integration sequence of four maizes and maize endogenous reference gene Zein which come from NCBI databases, five primer pairs and five probes with high specificity were analysed, designed and screened for each sequence using DNA star、Primer5.0、DNA MAN and Primer Express3.0. Specific gene probes of all these five target gene labeled with aminolinker C-12were prepared and coupled with fluorescencecoded microspheres. Biotinylated PCR amplicons were then hybridized with the probes. And then the liquichip detection technique for detection of all these five target gene was established by using Bio-plex200System to detect fluorescence signals in the reaction system. And five kinds of specific genes cloned plasmid DNA were constructed, and were identified by sequencing. The comparison between the sequencing results with the NCBI sequence are close to100%separately, which ensure accurately to do the follow-up test. The method was optimized by the ratio of sense and antisense primers, PCR produced products of the hybrid system, hybridization temperature and hybridization time. The rapidly high throughput liquichip detection technique to detect the four GM maize lines was established. The method is rapid, and the time of entire experiment is within2hours. The results indicated there were no cross reactions among five probes. The detecting limitation for the GM maize lines could reach to0.01%by detecting the different content of genetically modified maize DNA, so the method have highly sensitive. The repeatability test indicated that coefficient of variation is less than8%.We developed a liquid chip detection method for detecting four Genetically Modified Maize, which can detection directly four strains of genetically modified corn, and ultimately determine the sample whether containing the target strains. This study could make the technical reserve ready for the detection of the genetically modified maize. And provide lessons experiences and a good testing platform for other rapid high-throughput detection of genetically modified crops as corn, soybeans and rice et al. |
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