Molecular detection primer evaluation and absolute quantitative PCR system optimization of Candidatus Liberibacter asiaticus(CLas)
Citrus Huanglongbing(HLB)is the most devastating disease for the citrus industry.Currently,there are no effective cure and disease-resistant varieties available.Molecular detection is crucial for the effective prevention and management of Huanglongbing.In this study,we evaluated the commonly and world-widely employed primers for conventional PCR and nested PCR detection,and constructed plasmid standards based on multiple-copy nrdB and 16S rDNA genes,and then screened the best plasmids suitable for absolute quantitative PCR.The results showed that among the 16 pairs of primers evaluated in this study,OI1/OI2c,Las606/LSS and HLBF468/R877 had the highest sensitivity when using Es Taq MasterMix for conventional PCR detection of Candidatus Liberibacter asiaticus(CLas)infecting citrus samples,which was recommended to detect samples with low CLas titer.Each nested PCR detection primer had their applicable amplification systems,and some primers showed strong non-specific amplification when amplified with Es Taq MasterMix,while F1/B1→F3/B3 were suitable for Es Taq MasterMix system,and could stably and specifically detect the 105-fold diluted citrus total DNA samples(2×10-3 ng/μL)infected with CLas,which was the primer set with the highest sensitivity.OI1/OI2c→S3/S4 could stably and specifically detect 104-fold diluted citrus total DNA samples(2×10-2 ng/μL)infected with CLas both in Es Taq MasterMix and Ex Taq DNA polymerase systems,which was the primer set with the widest applicable amplification systems.Among the five plasmid standards constructed for absolute quantitative PCR,pnrdB83 was the best plasmid whose amplification efficiency was closest to 100％and with the least fluctuation and strongest stability between two repeated experiments.Besides,when used pnrdB83 as a standard for absolute quantification of Huanglongbing samples,the copy number difference of each sample between the two repeated experiments is the smallest.The results will provide a reference for qualitative and quantitative molecular detection of CLas.
万方数据
维普
