Development And Comparison Of Rapid Detection Method Technology For Candidatus Liberibacter Solanacearum

Potato zebra chip,caused by the fastidious Gram-negative bacteria Candidatus Liberibacter solanacearum(Lso),is a destructive disease in the world and leads to huge economic losses,quality deterioration and plant death.So far,Potato zebra chip(referred to as PZC)disease has been widely spread among 6 continents in the world.In 2014,PZC disease and and its vector insect potato psyllid were listed as an A1 quarantine pest by European and Mediterranean Plant Protection Organization(EPPO).It is also classified as an entry quarantine pest of the People’s Republic of China.The key to controlling PZC disease is to improve the detection methods of pathogenic bacteria.At present,the detection methods of Lso mainly include microscope observation,conventional PCR,nested PCR,multiplex PCR,real-time fluorescence quantitative PCR,etc.However,these detection methods all have certain limitations,and still need to develop an efficient detection method with strong reliability,strong repeatability,high sensitivity,low cost and short detection cycle.Therefore,in this paper,investigations on Lso detection in the imported seeds were carried out,and established the detection system of the internal amplification control(IAC-PCR),a recombinase polymerase isothermal amplification nucleic acid(RPA)system and a droplet digital PCR(dd PCR)system.Lso in importing seeds was detected by the above three methods,and the following research results were obtained:(1)Established the detection system of IAC-PCR for Candidatus Liberibacter solanacearum:According to the ACTB gene of pig as a internal amplification control,a compound primer recombination method was used to construct an amplified internal standard plasmid,andthe specific primer of Lso TX 16/23F/Lso TX 16/23R from EPPO standard was selected.A PCR detection method for the Lso containing amplified internal control was established through optimized the amount of IAC added.The results showed that when the amount of amplified internal control was 3.13×10~2copies,the detection sensitivity was not affected,and the amplification effect was the best.The detection sensitivity of the Lso plasmid was 4.05×10~4copies/μL.(2)Established the detection system of RPA for Candidatus Liberibacter solanacearum:Based on Ribonucleotide reductase(RNR)β-subunit gene of Lso,six pairs of primers were designed to establish RPA detection method.The results showed that the specificity of nrd B F4/nrd B R4 primer was the strongest,and the plasmid detection sensitivity was 1.22×10~2copies/μL,which was about 2 orders of magnitude higher than the detection limit of conventional PCR.(3)Established the detection system of dd PCR for Candidatus Liberibacter solanacearum:The dd PCR system of Lso was established using the EPPO specific fluorescent primer Lso F/HLB/HLBP.The results showed that the optimal annealing temperature was 58℃;the one-dimensional scatter diagram of linear plasmids was more concentrated than that of circular plasmids.The predicted concentration was positively correlated with the actual quantitative concentration,showing good linear relationship.The detection limit for plasmid was 0.51 copies/μL,two orders of magnitude higher than that of the q PCR method.Dd PCR showed good repeatability,with the minimum value of variation coefficient of 5.39%and the maximum value of 14.05%,both of which were less than 15%.(4)In the actual detection of 85 batches of seed samples,the positive detection rates of IAC-PCR,RPA and dd PCR were 15.00%,26.25%and 31.76%,respectively.Among the three detection methods of Lso,the detection sensitivity was IAC-PCR<RPA<dd PCR.(5)By comprehensive comparison of the advantages and disadvantages from IAC-PCR,RPA and dd PCR,we suggested that RPA was suitable for the promotion of testing laboratories at grassroots ports.