The Study Of DNA Detection Based Internal Marker Tracer Method For Food Traceability

Since food safety relates to life and health of people directly, it has always been ahot social issue. For that the phenomenon of food adulteration and counterfeiting isvery severe, the establishment of the food safety traceability system is not perfect andthere are many defects in the application of this system, a new concept of the internalmarkers trace method was put forward by many researchers, the main idea of whichis making special material as internal markers adding into the goods for tracing,anti-counterfeiting and authentication. Nucleic acids become the first option of theinternal markers owing to its advantages such as sequence diversity, high security,stable physical and chemical properties and strong specificity. A variety of DNAamplification techniques can be used to test the invisible barcode aiming at thesource and authenticity of goods. In order to make up for the deficiencies oftraditional anti-counterfeiting and authentication techniques, as well as theapplications of traceability system, a new internal markers method based on avariety of DNA amplification techniques for liquid food traceability and identificationis established in this study. At the same time, this study also provides more reliabletheories about the practical application of this method, which has a wide range ofapplications.Only the nucleic acids are stable in the liquid food, can it have the qualify to bethe internal markers. Firstly, the PCR and rolling circles amplification (RCA)technology was used to test the stability of double-stranded and single-stranded DNArespectively, which are stored in nine common liquid foods. The results show thatdsDNA and ssDNA are stable in most liquid foods, which provides a reliable theoriesfor the practical application. Meanwhile, the internal markers trace method basedon PCR and RCA techniques in the traceability, anti-counterfeiting and authentication of liquid food is established. The limit of detection (LOD) of the PCR and RCAtechniques is10fmol/L and10pmol/L, respectively.A new efficient primer-free DNA amplification technique is put forwardaccording to the expansion characteristics of the repetitive sequences and the researchof the ab initio DNA synthesis in this thesis. The main principle of this amplificationtechnique is: palindromic sequence in the end of DNA strand can form the hairpinstructure, the3’-end of which can be used as primer to lead the extension of the DNA,so40-50nt oligonucleotides can be used as templates directly in this method. Theresults show that this method performed well in a simple system of water as well asthe Chinese spirit, the LOD of which is100pmol/L. In order to improve the LOD,the enzyme is added into the reaction system to improve the amplification efficiency.The theory of the improved method is based on the Cut-grow amplification model inthe process of ab initio DNA synthesis. The results show that the LOD can be1fmol/L after improvement, but other nonspecific amplification may occur leading tothe false detection result. More research was needed to solve this defect. In brief, itcan be predicted that primer-free DNA amplification technique will have broadapplication prospects in the respects of food anti-counterfeiting and traceability for itsgood stabilities and simple operation.The thermodynamic stability of special stem-loop structures that contain d(cGCAg) or d(cGAAAg) are tested by measuring their Tm. A similar primer-freeamplification method is designed based on the combination of both special stem-loopstructures and efficient primer-free DNA amplification technique. The main principleof this amplification method is: special stem-loop structures can be formed at the endof the DNA strand,3’-end of which can be used as primer to lead the extension of theDNA. The results show that the oligonucleotides that have special stem-loop structurecan elongate, which provides some references for the application of the specialstem-loop structures and other DNA amplification techniques.In conclusion, it’s possible that the application of internal standard substancetrace method based on a variety of nucleic acid amplification techniques in the traceability, anti-counterfeiting and authentication of liquid food.and the method willhave broad application prospects in the future.