Optimization of the annealing temperature specific primers for detection of phytase gene from Rhodotorula mucilaginosa RG-PK20

Abstract

Phytic acid is an anti-nutritional substance that can reduce digestibility, nutrient absorption, and the efficiency of feed utilization in livestock. Phytase is an enzyme capable of hydrolyzing phytic acid into inositol and phosphoric acid so that it can increase the absorption of nutrients in the digestive system. Phytase can be found in microorganisms such as molds, yeast, and bacteria. Rhodotorula mucilaginosa is one of the reported yeasts that has the potential to produce phytase enzymes. The aim of this study was to determine the optimal annealing temperature for detection the phytase gene in the Rhodotorula mucilaginosa RG-PK20 genome using several pairs of specific primers. This research was initiated by designing specific primers for phytase genes through the online site https://www.ncbi.nlm.nih.gov. Comparative sequence analysis using Galaxy (https://usegalaxy.org/). Optimization of the primary annealing temperature (Ta) using the PCR method. Four pairs of primers were used, where two pairs of primers (FitAF/FitAR and FitaseF/FitaseR) were designed from research results and two pairs of primers had been validated from previous studies. The amplification results of the design primers FitAF/FitAR and FitaseF/FitaseR on Rhodotorula mucilaginosa RG-PK20 genomic DNA were optimal at all temperatures (52, 54, 56, 58, and 60 °C), with the formation of DNA bands with size of ± 500 bp. Detection using PhyR/F primers resulted in specific DNA bands with a size of ± 1171 bp at optimal temperatures (Ta) of 56, 58, and 60 °C. However, in contrast to the FiF/FiR primers pair, it was not specific to the phytase target gene.