| Bacterial fruit blotch of watermelon caused by Acidovorax avenae subsp. citrulli (Aac) isone of important national plant quarantine disease in China. Aac mainly infects watermelonsor other melons which belong to Cucurbitaceous crops. It has become the most seriousdiseases threatening the watermelon production and it can incite devastating disease epidemicswith the favorable environmental conditions. Contaminated watermelon seed is the primarysource of the disease spreading. Until now,the most efficient disease-management strategy isto eliminate the infected seeds prior the planting. However, the relatively low proportion ofinfected-seed makes the disease management especially difficult. Other traditional detectionmethods require extra time, labor intensive but also need the knowledge of the pathogeniccharacteristic and the disease symptoms. Therefore, it is necessary to develop accurate, rapidand sensitive methods for detecting Aac invasion and preventing of the spread of disease.The objective of this study was to explore and develop molecular detection techniques forAac.The results showed as below:1. A series dilution of bacterial suspension and seed extracts suspension were boiled torelease DNA for the Bio-PCR,MF-PCR,IMS-PCR,IC-PCR,Direct-PCR and DAS-ELISAanalysis. Comprehensive comparisons were made based on the sensitivity, stability, time andcost. The data showed that both IC-PCR and IMS-PCR are considerable higher sensitivitymethods, the sensitivity of IC-PCR could reach4.7×10~2cfu/mL and that of IMS could reach4.7×10~2cfu/mL~4.7×10~3cfu/mL. Meanwhile, the true positive detection rate was66.7%in0.1%rate of infected-seed. Although the procedure time was longer than Direct-PCR (4h) andMF-PCR(5h), it finished within12h. The expenses of the detection those two methods wasalso very low. Therefore, our results suggested that IC-PCR and IMS-PCR are sensitive,specific, rapid, reproducible, and economical methods for detecting Aac in watermelon seeds.2. The specific primer set BX-L1/BX-R5and BX-L1/BX-S-R2derived from BOXPCRwere selected to develop a nested PCR in single assay of Aac. The optimal PCR reactionsystem was50μL:7.5μL10PCR buffe(r25mM MgCl2),4μL dNTP(D4030RA,2.5mM),3μL primer(5μM/L),0.25μL Taq(DR001B,5U/μL). the direct pyrolysis was used to releaseDNA as template in this system, the amplified result was clear.3. The DNA of Aac suspension, mocked seeds, and different carrier rate of seeds wereamplified by50μL system of nested-PCR, it was indicated that the lowest detection limit canreach2.4×10~1cfu/mL, and the positive detection rate was100%in0.1%of infected-seed.4. Setting cellulose nitrate film as vector and bacterial fruit blotch of watermelon as amodel pathogen, the specificity of the capture antibody was arrayed as an array pattern samplefor arrangement and fixation; we established an antibody microarray detection method. Themain parameters of sample concentration, incubation condition, hybridization reactionconditions, chromogenic time were optimized, and a visible protein chip was established. 5. Using the visible antibody microarray detection technology, the suspension of Aac,mocked seeds, and infected-seed with varies infection were analyzed. My results showed that:the lowest detection capability reached2.4×10~4cfu/mL~2.4×10~5cfu/mL. In the proteinchip system, only1/25dosage of the capture antibody and1/8of the detection time couldreach the same detection capability that achieved by ELISA. In addition, the cost of detectionwas low and the procedure was simple and convenient. |
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