Detection Of Citrus Huanglongbing Based On Nucleic Acid Amplification Technique

Citrus huanglongbing(HLB)is one of the most destructive diseases of citrus worldwide,which causes huge economic losses to the citrus industry.The implementation of its rapid field detection has great significance.Las(Candidatus Liberibacter asiaticus)was chosen as the research object in this project for it was the main pathogenic bacteria of HLB.Focusing on the problems existing in field detection,we chose nucleic acid amplification technology and evaluated its three modules of nucleic acid extraction,nucleic acid amplification,and amplicons detection to be more rapid and simpler,to exploring novel,simple and rapid field detection methods for Las.Main contents and results are as followed:(1)A real-time PCR detection method for Las was developed.Three sets of PCR primers were designed for Las and one of them which had the best performance was selected for the following assay.The nonspecific amplification of PCR was inhibited by adjusting the annealing temperature.The sensitivity of this real-time PCR was confirmed to be equal to the widely used OI1/OI2c PCR.In conclusion,this developed real-time PCR can replace OI1/OI2c PCR for the daily detection of Las.(2)A LAMP-based visual field detection method for Las was developed.A set of LAMP primers was designed and LAMP system was optimized so that it could be completed within 35 min.Through experiment exploration,the optimal conditions for NaOH-based DNA crude extraction was confirmed to be 3 min treatment and 50-fold dilution.Then,a novel visual detection method for LAMP amplicons based on low concentration fluorescent dye was developed.And a mini fluorescent-emission cartridge was designed for this method.It was found that the background signal of visual detection came from the relatively long and high concentration LAMP primers which was easy to form secondary structure.Fortunately,the background signal could be eliminated by heating treatment.The sensitivity of this LAMP-based visual field detection method was confirmed to be about 100 times more sensitive than conventional TaqMan PCR.Its reliability was also verified by analyzing practical samples.In conclusion,this developed LAMP-based visual field detection method for Las is simple,rapid,sensitive,easy to judge and needing no complex instruments,making it suitable for the field detection of Las.(3)An RPA-based visual and rapid field detection method for Las was developed.Three RPA primer pairs were designed for Las and one of them which had the best performance was selected.The reaction temperature was optimed as 37 ?.The best duration time of NaOH-based DNA crude extraction for RPA was confirmed to be 1 min.It was found that the optimal reaction condition to eliminate the background signal of SYBR Green I-based visual detection method for RPA amplificons was 2-fold diluting the reaction mixture after 10 min amplification with 0.36 ?M primers.A disposable inclosed cartridge was employed to prevent carryover contamination of visual detection.The sensitivity of this developed method for Las was confirmed to be equal to the widely used OI1/OI2c PCR.And its reliability was also verified by analyzing practical samples.In conclusion,this RPA-based visual and rapid field detection method for Las is simpler,more rapid,and more cost-friendly than LFD method previously reported.The whole detection process can be conducted within 15 min and needs no complex instruments,making it a good choice for resource-limited areas and field detection of Las.