Optimization of the Detection Method for Xanthomonas axonopodis on Chili Pepper Seeds Using Polymerase Chain Reaction with Two Difference Primers

Abstract

The detection of Xanthomonas axonopodis pv vesicatoria (XAV) from chili seeds was carried out to avoid spreading XAV through seed. Detection of XAV in chili seeds through Polymerase Chain Reaction (PCR) using XAVF/XAVR and RST65/RST69 primers sometimes got unclear electrophoresis results. This research was conducted to determine the optimal time and temperature of primer annealing, and primer concentration to detect XAV on chili pepper seeds. Optimization was conducted in three phases, (1) optimization of primer annealing temperature, (2) optimization of annealing time, and (3) determinate primer concentration. The results show that XAVF/XAVR primer is optimal at 60°C for 30 seconds on the primer concentration of 10 pmol, and primer RST65/RST69 is optimal at 58°C for 20 seconds on the primer concentration of 10 pmol. The optimal time, temperature of primer annealing, and primer concentration were validated by three repetitions using positive and negative control. The validation test result shows that optimal time and temperature of primer annealing, and primer concentration consistently produce clear electrophoresis results without smear.