Optimization of PCR Detection Technology for Citrus Huanglongbing
In order to improve the efficiency and detection rate of citrus Huanglongbing detection system,the author combined the amplification system established by Jagoueix et al.to optimize DNA extraction,PCR system,and PCR program.This study used different extraction times,different cracking temperatures,and different combinations of antioxidants to study the impact on DNA quality,as well as by setting primer concentration gradients and annealing temperature gradients to study their impact on detection performance.The results showed that the extraction frequency had little effect on DNA quality.The quality of DNA extracted by water bath lysis at 65℃was better than that by room temperature lysis,but the overall difference was not significant.Adding to DNA extraction solution β-Mercaptoethanol β-Mercaptoethanol and PVP,both of which were not added,had little effect on the quality and purity of the final total DNA.Stable characteristic bands appeared at primer concentrations of 0.4-1.0 μmol/L,and primer dimers were still formed at primer concentrations of 0.1 μmol/L The detection results were optimal at an annealing temperature of 66.5℃.Therefore,adopting a single extraction,room temperature lysis,and no addition of antioxidants can simplify the DNA extraction process.Adjust the primer concentration to 0.8 μmol/L had the best effect.Annealing temperature of 66.5℃could improve the detection rate.The optimized detection system was easy to operate,stable,and had a high detection rate,making it suitable for batch detection of Huanglongbing.
Citrus HuanglongbingDNA extractionPCRdetection system
国家科技期刊平台
NSTL
万方数据
