ISSN 2756-3340
African Journal of Infectious Diseases Research ISSN 4729-6836 Vol. 4 (11), pp. 001-009, November, 2017. © International Scholars Journals
Full Length Research Paper
Comparison of pathogen enrichment methods for detecting Acidovorax avenae subsp. citrulli” from watermelon seeds
WANG Jing1, LIU Qing2, CHEN Nian-lai3, LIU Jin-zhi4, and BI Yang1*
1College of Food Science and Engineering, Gansu Agricultural University, Lanzhou 730070, P.R. China
2Gansu Entry-Exit Inspection and Quarantine Bureau, Lanzhou, 730020, P.R. China.
3College of Resources and Environment, Gansu Agricultural University, Lanzhou 730070, P.R. China.
4College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, P.R. China.
Accepted 18 May, 2017
Abstract
It is well known that bacterial fruit blotch (BFB) of watermelon caused by Acidovorax avenae subsp. citrulli threatens watermelon production. There is no pathogen enrichment or concentrated steps before detection in conventional PCR-based diagnostic technique, which consequently leads to the lower detection sensitivity. In the present study, two different bacterial suspensions were prepared, and four pathogen enrichment protocols including Biological Preparation (BP), Membrane Filtration (MF), Immune-Magnetic Separation (IMS) and Immune-Capture Preparation (ICP) were employed to prepare the PCR template for the pathogen detection. Double Antibody Sandwich Enzyme Linked Immunosorbent Assay (DAS-ELISA) was also conducted as a parallel comparison. The results showed that IC-PCR was the optimum method through comparing the detection limits, time and expenditure among those methods. The detection limit of IC-PCR for both bacterial suspension and seeds suspension can reach 102 CFU/ml, which was 10 times lower than that of IMS-PCR when the seed suspension was used as template. In addition, the results of IC-PCR showed higher degree of precision than those of IMS-PCR. The time and expenditure of IC-PCR were comparable with other methods. The present study demonstrated that the procedures of immune-capture PCR is a sensitive, specific, rapid, reproducible, and economical method to detect A. avenae subsp. citrulli in watermelon seeds.
Key words: Bacterial Fruit Blotch, watermelon seed, pathogen enrichment, PCR.