Refinement of Polymerase Chain Reaction (PCR) Process for GM Papaya Detection in Small Laboratory

Abstract

The ban of Genetically Modified crops importation by many countries, had papaya exporters inThailandscreeningforgenetically modified(GM)papayathroughout theproduction chain i.e., seedling selection, raw material purchasing, and before product exporting to prevent theproduct from beingrejectedat thereceivingend. Nowadays, Real-time PCR technique is popular for the GMO screening and recognized as an international standard method but is high cost to run and high investment for equipment. The aim of this study was to refine the PCR technique that was a basic technique for GM papaya detection in small laboratories. The internal factors (annealing temperature, primer set, DNA concentration and DNA extraction methods) and external factors (PCR machine brand, PCR programs and PCR cycles) for GM screening: the CaMV35S promoter, Nos terminator, neomycin phosphotransferase (nptII), and papain gene by PCR technique were improved. The studies revealed that GM screening by PCR technique have to consist of the following factors:1) The optimum annealing temperature was 58๐ C. 2) The primer final concentration was 0.2 uM and 3) the optimum initial DNA content was 50 nanograms per reaction. Improper DNA extraction methods that did not remove all inhibitorsaffect the PCR reactionyield. Inaddition, reducingtheinitialdenaturationandextension steps by 5 min each and reducing the number of PCR cycles by 10 can help reduce the GM papaya detection processing time by 40 min. and be as effective as the original program that takes approximately 2 hrs.