Detection methods for genetically modified foods

　　In 1996, Meyer Rolf et al. of the University of Bernstein in Germany demonstrated the possibility of PCR detection of genetically modified foods. The genetic components transferred into plant cells generally include promoters, reporter genes, target genes and terminators, of which promoters and terminators are necessary for the expression of target genes. At present, the promoters used in transgenic plants are mainly derived from the Camv35s promoter of cauliflower mosaic virus, the nos promoter of Agrobacterium tumefaciens and the FMV35s promoter of figwort mosaic virus; the widely used terminators are derived from the nos terminator of Agrobacterium tumefaciens, the camv terminator of cauliflower mosaic virus and the neomycin phosphotransferase NptII terminator. PCR amplification of these genes can cover 95% of the needs of existing transgenic plants.

　　PCR qualitative testing is a highly sensitive DNA level test, but it is often accompanied by various false results: (1) Various reaction inhibitors produced during DNA extraction cause PCR to be false negative; (2) During deep processing of products, nucleic acids are broken into fragments and the content is extremely low, causing PCR to be false negative; (3) When crops are infected with cauliflower mosaic virus and carry the 35S promoter or infected with Agrobacterium and carry the nos terminator, PCR will be false positive; (4) Residual contamination in the laboratory causes the test result to be false positive; (5) Cross-contamination between genetically modified foods and non-genetically modified foods during harvesting, transportation or processing will also cause inaccurate test results.

　　To prevent false negative results in the test, it is necessary to strictly adhere to the operating procedures during the test, optimize the PCR conditions, try to remove substances that may inhibit the PCR reaction during the DNA extraction process, and eliminate false negatives by amplifying the 18SrRNA gene of eukaryotes. To eliminate the impact of false positive results in genetically modified food testing, methods such as southern hybridization, PCR product purification sequencing, or PCR amplification product enzyme analysis can be used to simultaneously detect the Camv35s and nos gene double elements of the sample, which can avoid false positive results caused by natural infection of certain crops with cauliflower mosaic virus or Agrobacterium tumefaciens.

　　Real-time fluorescence quantitative PCR

　　Real-time fluorescence PCR technology was introduced by Applied Biosystems in the United States in 1996. It refers to the addition of a probe labeled with two fluorescent groups on the basis of conventional PCR, using the accumulation of fluorescent signals to monitor the entire PCR process in real time, and finally using the standard curve to quantify the unknown template. At present, there are three main types of fluorescent probes used in real-time quantitative PCR detection systems: molecular beacon probes, TaqMan probes, and hybridization double probes.

　　The sensitivity of real-time quantitative PCR detection is at least 10 times that of competitive PCR, and it can detect 2pg of transgenic DNA per gram of sample. It can detect processed, unprocessed and mixed samples.

　　Real-time fluorescence PCR technology effectively solves the limitation of traditional quantitative end-point detection , and detects the intensity of the fluorescence signal once in each cycle, and records it in the computer software, which can monitor the entire PCR process in real time. In addition, real-time fluorescence PCR uses closed-tube analysis, without the need for post-PCR processing steps such as electrophoresis, and can effectively eliminate cross-contamination of nucleic acids. Since fluorescent probes are added to the PCR reaction system, non-specific products cannot hybridize with the probes, which is especially effective for products with molecular weights close to those of specific products that cannot be separated by electrophoresis. The current challenge of this method is the lagging development of quantitative standards, and commercial standards can no longer meet the growing needs of transgenic detection. Moreano et al. developed a new method for synthesizing standards with transgenic rapeseed as the detection target, thus pointing out the direction for improving the standardization of fluorescence quantitative detection.

　　Gene chip detection method

　　The most advanced technology used in the detection of genetically modified foods today is gene chip technology, which is essentially a highly integrated reverse dot hybridization technology. The probe molecule is fixed on a carrier, and the gene to be tested undergoes PCR, end labeling and other operations to become a nucleic acid molecule labeled with a fluorescent dye or isotope, which is then hybridized with the fixed probe. Depending on the labeling method, the intensity of each spot signal is read out by radioautography, laser confocal microscopy or CCD camera, and the computer processes the hybridization signal to obtain the hybridization spectrum. This technology was first developed by Affymetrix in the United States in the 1990s, and the company produced the world's first commercial gene chip in 1996.

　　In most cases, immunology and PCR detection technology can only detect one target molecule in one experiment, and in a few cases, it can detect 2 to 3 target molecules at the same time. Gene chips can solve the problem of large-scale gene detection, and have the advantages of high sensitivity, high efficiency, low cost, automation, and clear results. However, due to the high cost of the corresponding equipment required for its application, the narrow scope of popularization, and the lack of any standards, the application scope of gene chip detection is not wide.

　　Multiplex ligation-dependent probe amplification (MLPA)

　　This method is the latest development in the multiple quantitative detection of transgenic genes. It was originally published by Dr. Schouten JP of the Netherlands in 2002 as a highly sensitive relative quantitative technology for medical detection purposes. It uses simple hybridization, ligation, and PCR amplification reactions to simultaneously detect the copy number changes of up to 40 different nucleotide sequences in a single reaction tube.

　　The MLPA method is a detection method that designs multiple sets of specific probe groups for different detection sequences and amplifies the probe groups. Each probe group has a different total length and can hybridize and bond with the target sequence. All probes have a universal primer binding region PBS (primer binding sites) at the 5′ end and a binding region for the target sequence to be amplified at the 3′ end. Oligonucleotides of different lengths are inserted between the PBS region and the target sequence binding region to form probe groups of different lengths. If the target sequence is missing, mutated, or due to the pairing of different probe groups, this set of probes cannot be successfully connected and there is no corresponding amplification reaction. If this set of probes can completely bond with the target sequence, the ligase will connect this set of probes into a fragment and amplify the connected probe group through a labeled universal primer. Finally, the amplified product is detected by capillary electrophoresis and laser-induced fluorescence.

　　Francisco Moreano et al. applied this method to detect standard transgenic soybeans and corn and verified the feasibility of MLPA in transgenic detection through specificity and sensitivity experiments. The experiment showed that the longer the fragment length of the probe binding to the target sequence, the higher the sensitivity of the detection, and based on the linear relationship between the ratio of the transgenic sequence amplification intensity to the internal reference gene amplification intensity and the transgenic content, the sample to be tested can be quantitatively analyzed.