Justin Godfred B.

Peralta FST 202 B-1L Recent Advances on DNA based method in Food Analysis

Food Analysis From the introduction of the application of science in the study of food and consumption, it has been observed that analysis played a key role in the Food industry. From determining composition to the presence of adulterants, food analytical methods are essential in maintaining quality and integrity of food products for consumer use. The Speed and Accuracy of the results are crucial in choosing a method for analysis. Methods which are rapid and accurate are favored over those which lack one of the two. Among these methods, one of the methods that are known to possess these attributes is through DNA-based methods of analysis. DNA Analysis DNA Analysis is a known method for the determining the genetic make-up of a substance of a biological substance. It is based on the concept that the genetic composition of a given material is unique to that material and is a good basis for its identity (i.e. Beef contains genetic components attributed to beef, not for pork or chicken). Essentially, the use of DNA Analysis is able to detect the presence or absence of a particular component of food. This pertains to the use of immunoassays such as ELISA paired with DNA amplification methods such as Polymerase Chain Reaction or PCR. A combination of the two methods results in a method of analysis known as DNA immunoassay.

Polymerase Chain Reaction PCR is a method for DNA amplification which utilizes the initial ability of DNA polymerase enzyme to synthesize DNA strands. The process involves obtaining a target sequence of the DNA and amplifying it through multiple copies. This entails a qualitative result in determining the coding sequence for a specific substrate. Many forms of PCR are utilized in food analysis particularly in allergen detection. These methods include PCR-ELISA, Real-time PCR and PCR-PNA-HPLC. PCR-ELISA Immunoassay Immunoassays focus on the immunological biochemical reactions that occur with respect to particular components. The use of antibodies which react with a specific analyte of interest, are implored in detection and quantification of substances particularly those which are bio-molecular in structure (i.e. proteins, peptide chains and microorganisms). The most common Immunoassay is known as Enzyme-linked immunoabsorbent Assay (ELISA) There are 2 main components for involved with ELISA, capture and detection The differences in method of attachment as well as detection of the biomolecule create the variation of the technique. In Direct capture ELISA, the desired component, known as the antigen, is bound to the wells of the plate used for analyses while in indirect capture, the antigen binds to a specific antibody attached

into the well. The detection methods are also differentiated through direct and indirect methods. Direct detection includes the use of another antibody with a detection enzyme which binds to the antigen. The detection enzyme is quantified through the use of signaldetection such as spectrophotometry. Indirect detection involves the use of two additional antibodies, a primary antibody, one which binds to the other end of the bound antigen and a secondary antibody which binds to the primary antibody. The secondary antibody contains the detection enzyme which contains the signal. Comparatively, the indirect detection is preferred due to its ability to produce stronger signals. This is due to the ability of multiple secondary antibodies to bind with the primary antibody. A commonly used combination is the sandwich ELISA which makes use of an indirect method of capture and an indirect method of detection. Another mode of ELISA, known as Competitive ELISA involves the use of the labeled antigen as the quantifier. Through indirect capture, the sample with the desired antigen is inoculated with labeled antigens and primary antibodies which compete with the antigens in the sample in binding with the antibody and bind to the bound antibodies respectively. Since the binding method is concentration dependent, a high antigen concentration would generate a low signal and vice versa (Hayworth, 2014). In the early use of the technology, the use of DNA-based antigens is minimal in the food industry and is more utilized in medicine for viral detection. Recent advancements in Immunoassay technology have been able to show more applications in the food industry through the use of PCR-mediated DNA amplification. The DNA fragment amplified through PCR is then labeled with a detector

enzyme and is then analyzed through ELISA methods such as competitive ELISA. This entails a faster analysis since the conventional analysis of electrophoresis requires a longer and more time-consuming procedure. This method of detection is normally applied for monitoring of allergens in trace amounts. Real-Time PCR Quantitative PCR or Real-time PCR is a modification of the basic PCR method in a way that monitoring occurs during analysis. This allows the concentration of the gene to be traced from the start of the analysis up to the end. This allows detection during reaction of synthesis. This allows a more in-depth monitoring of the amount of sample synthesized as well as how much was detected prior to analysis. There are two methods of detection during PCR: intercalation or inclusion of a non-specific fluorescent dye within the DNA strand thus making it detectable through spectroscopy and addition of DNA probes which bind to specific areas of the DNA. The Probes are only activated when the specific protein sequence is achieved. This method is also applied in the field of allergen detection. PCR-PNA-HPLC PCR-PNA-HPLC technique is a modification of the basic PCR wherein biomolecular probes are attached to the DNA and are detected through HPLC. This ensures a more in depth and sensitive process and component analysis. It also ensures a more rapid output of results with increased detection potency. This allows detection of some allergenic foods such as hazelnut in some vegetable products which may be undeclared or as possible contamination (Slowianek & Majak, 2011).

Other Potential Use and Methods Aside from allergen detection, there are other uses for PCR-mediated DNA Analysis on food. Speciation or the process of determining the species of the meat sample obtained utilizes RFLP probing or Restriction fragment length polymorphism probing. This uses the difference in DNA sequences after treatment with restriction endonucleases. Treatment leads to fragments of different lengths which are measured and detected through gel electrophoresis. These fragments are specific and are differentiated per species thus leading to differentiation within dressed and prepared meat such as fish fillet (Moran, 2013) Food authenticity also implores the use of PCR-RFLP techniques in testing for adulteration in meat such as the presence of Horse-meat in Beef (Bioline , 2013). The use of FINS or Forensically Informative Nucleotide Sequencing is a method wherein DNA sequences which are amplified through PCR are identified and subjected to phylogenetic analysis as compared to a database. This allows the authentication of the purity of a meat samples which were obtained post-slaughter (Bartlett & Davidson, 1992). Advantages and Disadvantages Analytical Methods of DNA

leading to deformation which results in erroneous results. Essentially, the presence of the DNA for the potential allergen source signifies the presence of the specie-specific DNA, not the allergen itself. This may lead to false-positive or false-negative results. Hence, the method of analysis is not recommended for those which are heavily processed as well as those which contain high protein but low DNA content (Slowianek & Majak, 2011).

Bibliography
Bartlett, S., & Davidson, W. (1992). FINS (forensically informative nucleotide sequencing): a procedure for identifying the animal origin of biological specimens. Biotechniques , 518. Bioline . (2013, February 14). Horsegate: Horsemeat DNA testing in Beef Products. Retrieved March 19, 2014, from Bioline: The PCR Company Blog: http://thepcrcompany.wordpress.com/2013/02 /14/horsegate-horsemeat-dna-testing-in-beefproducts/ Hayworth, D. (2014). Overview of ELISA. Retrieved March 19, 2014, from ThermoScientific: http://www.piercenet.com/method/overviewelisa#elisaformats Moran, L. (2013, October 16). Advances in Food Authenticity Testing Using DN A Techniques. Retrieved March 19, 2014, from Public Analyst Scientific Services: https://www.ifst.org/documents/events/liz%20 moran%20ppt.pdf Slowianek, M., & Majak, I. (2011). Methods of allergen detection based on DNA Analysis. Biotechnology and Food Science , 39-44.

Essentially, DNA Analytical methods through PCR-mediated techniques are known for their rapid, efficient and accurate results. The major disadvantage for DNA Analytical methods are the dependence of the analyses on the structure of DNA. DNA structures can be easily destroyed in the events of processing